Compositions and method for treating cardiovascular disease

ABSTRACT

The invention pertains to methods of treating cardiovascular disease by modulating inflammatory and immunoregulatory responses associated with such pathological conditions. Embodiments of the invention provide methods for the treatment of cardiovascular disease in a subject having cardiovascular disease comprising administering an effective amount of one or more IL-17 antagonists, IL-18 antagonists, 4-1BB antagonists, CD30 antagonists, OX40 antagonists and/or CD39 alone or in any combination. This abstract is provided for the sole purpose of enabling the reader to quickly ascertain the subject matter of the technical disclosure and is not intended to be used to interpret or limit the scope or meaning of the claims.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119 of U.S.Provisional Application Serial No. 60/494,457, filed Aug. 12, 2003, andU.S. Provisional Application Serial No. 60/406,418, filed Aug. 28, 2002.

FIELD OF THE INVENTION

[0002] Embodiments of the invention pertain to compositions and methodsfor treating cardiovascular disease by modulating inflammatory andimmunoregulatory responses associated with cardiovascular disease.

BACKGROUND

[0003] Cardiovascular disease encompasses a number of disorders thataffect the muscle and/or blood vessels of the heart, peripheral bloodvessels, muscles and various organs. It is established in the art thatinflammatory and immunoregulatory processes are implicated in thepathogenesis of various forms of cardiovascular disease.

[0004] For example, inflammatory immune responses have been shown tocontribute to the pathogenesis of atherosclerosis. Elevated levels ofC-reactive protein (CRP) have been associated with up to an 8.6 foldincrease in the relative risk of symptomatic atherosclerosis (Biasucci,L., et al., Circulation 1999, 99:855-860). Elevated levels of CRP alsopredict patients that are at elevated risk of myocardial infarction (MI)or stroke, and it has also been associated with poor prognosis inunstable angina (Vorchheimer, D., et al., JAMA 2001, 286:2154-2156).Binding C1q CRP activates the classical complement pathway and may leadto direct myocardial damage, coronary smooth muscle or endothelial celldeath and subsequent atherosclerotic plaque rupture (Agrawal, A., etal., J Immunol 2001, 166:3998-4004). Furthermore, a recent studydemonstrated that elevated levels of CRP are able to identify patientsthat die of sudden cardiac death 9 years prior to the event (Albert, C.,et al., Circulation 2002, 105:2595-9). These studies also imply thatlong-term inflammatory exposure and elevated CRP levels may contributeto the progression of acute coronary syndromes (Buffon, A., et al., NEJM2002, 347:55-7). Activation of inflammatory cells resident within anatherosclerotic plaque may elaborate enzymes capable of degradingextracellular matrix and lead to plaque rupture. Alternatively, theseinflammatory cells may directly kill endothelial and smooth musclecells. A number of studies have demonstrated that patients with unstableangina have peripheral T-cells that make enhanced levels of interferongamma compared to patients with stable angina. Furthermore, thereappears to be clonal expansion of a CD4+CD28null T-cell population inthese patients, which appear to be cytotoxic and can kill endothelialcells, an effect enhanced by CRP (Nakajima, T., et al., Circulation2002, 105:570-5).

[0005] Cytokines are critical regulators of the T-helper 1 (Th1) and Th2T-cell responses. The Th1 response results in pro-inflammatory cytokinerelease characterized by macrophage activation and, if unopposed, mayresult in tissue damage. The Th2 response results in a humoral immuneresponse, B-cell activation and an allergic reaction (Neurath, M., etal., Nat Med 2002, 8:567-73). A number of Th1 type cytokines includingTNF, IL-6 and the chemokine MCP-1 are elevated in unstable angina (AJC2001, 88(8A):10K-15K). Recently, IL-18 has been found to be anindependent marker for an adverse outcome in patients diagnosed withacute coronary syndrome (Blankenberg, S., et al., Circulation 2002,106:24-30). Elevated levels of IL-18 have also been found to correlatewith ulcerated, symptomatic carotid artery lesions (Mallat, Z., et al.,Circulation 2001, 104:1598-603). In a mouse model of atheroscleroticplaque development in ApoE deficient mice, IL-18 was shown to accelerateand enhance plaque formation, and IL-18 binding protein enhanced smoothmuscle proliferation, which would promote plaque stability by increasingthe thickness of the cap, and reduced the number of infiltratingmacrophages and T-cells (Mallat, Z., et al., Circ Res. 2001, 89:E41-5).Studies such as these establish a sound basis for implicatinginflammatory and immunoregulatory responses in cardiovascular disease.

[0006] There is an unmet need in the art for treating cardiovasculardisease by targeting the immunopathology of the disease. Embodiments ofthe present invention address such needs by providing compositions andmethods for treating cardiovascular disease by modulating theinflammatory and immunoregulatory responses associated withcardiovascular disease.

SUMMARY OF THE INVENTION

[0007] Embodiments of the present invention provide compositions andmethods for the treatment of cardiovascular disease in a subject havingcardiovascular disease, comprising administering an effective amount ofone or more antagonists, such as IL-17 antagonists that inhibit thebinding of IL-17 to the IL-17 receptor, as well as antagonists thatprevent or diminish the activation of the IL-17 receptor; IL-18antagonists that inhibit the binding of IL-18 to the IL-18 receptor, aswell as antagonists that prevent or diminish the activation of the IL-18receptor; 4-1BB antagonists that inhibit the binding of 4-1BB ligand to4-1BB, as well as antagonists that prevent or diminish the activation of4-1BB ligand or 4-1BB; CD30 antagonists that inhibit the binding of CD30ligand to CD30, as well as antagonists that prevent or diminish theactivation of the CD30; OX40 antagonists that inhibit the binding ofOX40 ligand to OX40, as well as antagonists that prevent or diminish theactivation of OX40; and/or CD39 alone or in any combination.

[0008] Additional embodiments are described in detail below.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIGS. 1A and 1B depict IL-17 and IL-18 levels, respectively, inheart donor plasma in relation to three distinct groups: surviving donorrecipients, donor recipients that died in less than 48 hrs. and unuseddonors with an ejection fraction (EF) of less than 30%.

[0010]FIG. 2 shows relative abundance of IL-18 Receptor in patientshaving an ejection fraction (EF) of less than 30%, patients in end-stagefailure (ESF) and patients having an ejection fraction of greater than60% (i.e., normal myocardium).

[0011]FIGS. 3A through 3D show comparative levels of IL-17 and IL-18plasma levels among patients in various stages of cardiomyopathy. FIGS.3B and 3D further dissect the patient population into non-ischemic andischemic groups.

[0012]FIG. 4 illustrates the plasma levels of IL-17 (open bars) andIL-18 (closed bars) and 4-1BB (hatched bars) of control subjects ordonors with favorable recipient outcome (group A) or adverse recipientoutcome (group B) post-transplantation.

[0013]FIG. 5 illustrates the effect of administering IL-17 on heartchamber dimensions over time.

[0014]FIGS. 6A and 6B depict IL-17 and IL-18 plasma levels inIFN-γ^(−/−) mice immunized with cardiac myosin peptide that induces aninflammatory myocarditis and cardiomyopathy.

[0015] FIGS. 7A through 7C: FIG. 7A shows IL-17 levels in mice from theexperimental autoimmune myocarditis (EAM) model, wherein mice havinghistologically demonstrated cardiopathology (animal B) had higherexpression levels of IL-17 over negative controls (animals C and D).FIG. 7B shows that IL-17 release from T-cells obtained from mice withEAM mice was approximately 25 fold higher compared to control mice.T-cells were isolated from animals immunized with cardiac myosin andexposed to antigen presenting cells fed myosin over antigen presentingcells not exposed to myosin. FIG. 7C illustrates that T-cells from ananimal immunized with cardiac myosin and having histological evidence ofcardiopathology (animal B) released high levels of IL-17 in response toantigen-specific stimulation by peptide-pulsed antigen presenting cells.

[0016]FIGS. 8A and 8B show IL-18 levels and the ratio of IL-18 to IL-18Binding Protein, respectively, in patients stratified into three patientgroups: those having stable coronary artery disease (CAD), acutecoronary syndrome (ACS) with cardiac troponin I (cTnI) plasma levelsless than 0.4 ng/ml or acute coronary syndrome (ACS) with cardiactroponin I (cTnI) plasma levels greater than 0.4 ng/ml.

[0017]FIG. 9 illustrates that 4-1BB plasma levels were significantlyelevated in patients with heart failure compared to normal subjectsthereby suggesting activation of this system in human heart failure.

[0018]FIG. 10 represents a dose-response of Adriamycin® in wild type and4-1BBL−/− knock out mice. At 22.5 mg/kg and 25 mg/kg, the percentage ofAdriamycin®-induced cardiac dysfunction was decreased in 4-1BBL−/−. Athigh dosage (30 mg/kg), no difference was observed between wild type and4-1BBL−/− group.

[0019]FIG. 11 is a graph showing 4-1BB activating antibody (M6)administered 3, 6, 9 days after Adriamycin® treatment. The onset ofdysfunction was accelerated by M6 antibody, but the final penetrance wassimilar to control group.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Embodiments of the invention provide compositions and methods fortreating cardiovascular disease in a subject having cardiovasculardisease comprising administering an effective amount of one or moreIL-17 antagonists, IL-18 antagonists, 4-1BB antagonists, CD30antagonists, OX40 antagonists and/or CD39, alone or in any combination.

[0021] Cardiovascular disease, as defined herein, encompasses diseasesand disorders of the muscle and/or blood vessels of the heart, diseasesand disorders of the vascular system, and/or diseases and disorders oforgans and anatomical systems caused by the diseased condition of theheart and/or vasculature. Examples include, but are not limited to:inflammation of the heart and/or vasculature such as myocarditis,chronic autoimmune myocarditis, bacterial and viral myocarditis, as wellas infective endocarditis; heart failure; congestive heart failure;chronic heart failure; cachexia of heart failure; cardiomyopathy,including non-ischemic (dilated cardiomyopathy; idiopathic dilatedcardiomyopathy; cardiogenic shock, heart failure secondary toextracorporeal circulatory support (“post-pump syndrome”), heart failurefollowing ischemia/reperfusion injury, brain death associated heartfailure (as described in Owen et al., 1999 (Circulation. 1999 May18;99(19):2565-70)); hypertrophic cardiomyopathy; restrictivecardiomyopathy; non-ischemic systemic hypertension; valvular disease;arythmogenic right ventricular cardiomyopathy) and ischemic(atherogenesis; atherosclerosis; arteriosclerosis; peripheral vasculardisease; coronary artery disease; infarctions, including stroke,transient ischemic attacks and myocardial infarctions). Additionaldisease states encompassed by the definition of cardiovascular diseaseinclude: aneurysms; arteritis; angina; embolism; platelet-associatedischemic disorders; ischemia/reperfusion injury; restenosis; mitraland/or tricuspid regurgitation; mitral stenosis; silent myocardialischemia; Raynaud's phenomena; thrombosis; deep venous thrombosis;pulmonary embolism; thrombotic microangiopathies including thromboticthrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS),essential thrombocythemia, disseminated intravascular coagulation (DIC),and thrombosis and coagulopathies associated with exposure to a foreignor injured tissue surfacethrombophlebitis; vasculitis, includingKawasaki's vasculitis; Takayasu's arteritis; veno-occlusive disease,giant cell arteritis, Wegener's granulomatosis; Schoenlein-Henochpurpura, as well as cardiovascular disease arising from periodontalinfections by one or more oral pathogens, such as bacteria. The examplesof cardiovascular disease provided above are merely illustrative andprovided to aid those of skill in the art to appreciate the scope ofcardiovascular disease that may be treated using the compositions andmethods described herein. Of course, other cardiovascular diseaseconditions may exist that can be treated using the inventivecompositions and methods. Additional examples of cardiovascular diseaseand disorders associated with cardiovascular disease, as well ascomplications arising from the treatment of cardiovascular disease, areprovided in the section below pertaining to therapeutic indications.

[0022] An “antagonist,” as defined herein, is a molecule that partiallyor completely blocks the binding of two cognates thereby inhibiting thedownstream biological effects of the cognates' interaction. For example,an antagonist may block the binding of a ligand to its receptor, whichin turn reduces and/or prevents intracellular signalling via activatingthat receptor, which in turn reduces or prevents the downstreambiological effects of activating that receptor, such as but not limitedto, cell activation, proliferation, differentiation, cytokine release,up-regulation of genes, cell-surface expression of proteins, and thelike.

[0023] Activating or activation of a receptor is defined herein as theengagement of one or more intracellular signaling pathway(s) and thetransduction of intracellular signaling (i.e., signal transduction) inresponse to a molecule binding to a membrane-bound receptor, such as butnot limited to, a receptor:ligand interaction.

[0024] “Signal transduction,” as used herein, is the relaying of asignal by conversion from one physical or chemical form to another. Incell biology, the process by which a cell converts an extracellularsignal into a response.

[0025] Antagonists presented herein comprise soluble receptor molecules,ligands and/or binding proteins, including IL-17, IL-17 receptor(IL-17R), IL-18, IL-18 receptor (IL-18R), IL-18 binding protein(IL-18BP), CD30, CD30 ligand (CD30-L), 4-1BB, 4-1BB ligand (4-1BB-L),OX40, OX40 ligand (OX40-L) and CD39. Antagonists presented hereinfurther comprise antibodies, fusion proteins and peptibodies directedagainst one or more of the following: IL-17, IL-17R, IL-18, IL-18R,CD30, CD30-L, 4-1BB, 4-1BB-L, OX40 and/or OX40-L. Antagonists presentedherein further comprise small molecules, such as peptidomimetics andmimotopes, and the like, that antagonize the interaction between IL-17and IL-17R, IL-18 and IL-18R, 4-1BB and 4-1BB-L, CD30 and CD30-L and/orOX40 and OX40-L. Additional antagonists comprise antisenseoligonucleotides that specifically target and hybridize to the mRNA ofIL-17, IL-17R, IL-18, IL-18R, CD30, CD30-L, 4-1BB, 4-1BB-L, OX40 and/orOX40-L thereby preventing gene translation of their respective proteins.Further embodiments comprise gene silencing by RNA-interferencemolecules tailored to silence expression of IL-17, IL-17R, IL-18,IL-18R, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40 and/or OX40-L. More specificdefinitions and examples of particular antagonists are provided in thesections below.

[0026] A “peptibody” refers to molecules comprising an Fc domain and atleast one peptide. Such peptibodies may be multimers or dimers orfragments thereof, and they may be derivatized. Peptibodies aredescribed in greater detail in WO 99/25044 and WO 00/24782, which areincorporated herein by reference in their entirety. The peptide may befrom the amino acid sequence of IL-17, IL-17 receptor (IL-17R), IL-18,IL-18 receptor (IL-18R), IL-18 binding protein (IL-18BP), CD30, CD30ligand (CD30-L), 4-1BB, 4-1BB ligand (4-1BB-L), OX40, OX40 ligand(OX40-L) and/or CD39.

[0027] A “peptide,” as used herein refers to molecules of 1 to 40 aminoacids. Alternative embodiments comprise molecules of 5 to 20 aminoacids. Exemplary peptides may comprise portions of the extracellulardomain of naturally occurring molecules or comprise randomized sequencesof of IL-17, IL-17 receptor (IL-17R), IL-18, IL-18 receptor (IL-18R),IL-18 binding protein (IL-18BP), CD30, CD30 ligand (CD30-L), 4-1BB,4-1BB ligand (4-1BB-L), OX40, OX40 ligand (OX40-L) and/or CD39.

[0028] The term “randomized” as used to refer to peptide sequencesrefers to fully random sequences (e.g., selected by phage displaymethods or RNA-peptide screening) and sequences in which one or moreresidues of a naturally occurring molecule is replaced by an amino acidresidue not appearing in that position in the naturally occurringmolecule. Exemplary methods for identifying peptide sequences includephage display, E. coli display, ribosome display, RNA-peptide screening,chemical screening, and the like.

[0029] The term “Fc domain” encompasses native Fc and Fc variantmolecules and sequences as defined below. As with Fc variants and nativeFc's, the term “Fc domain” includes molecules in monomeric or multimericform, whether digested from whole antibody or produced by other means.

[0030] The term “native Fc” refers to molecule or sequence comprisingthe sequence of a non-antigen-binding fragment resulting from digestionof whole antibody, whether in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immunoglobulins, although IgG1 and IgG2 are preferred.Native Fc's are made up of monomeric polypeptides that may be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms.

[0031] The term “Fc variant” refers to a molecule or sequence that ismodified from a native Fc but still comprises a binding site for thesalvage receptor, FcRn. International applications WO 97/34631(published 25 Sep. 1997) and WO 96/32478 describe exemplary Fc variants,as well as interaction with the salvage receptor, and are herebyincorporated by reference in their entirety. Thus, the term “Fc variant”comprises a molecule or sequence that is humanized from a non-humannative Fc. Furthermore, a native Fc comprises sites that may be removedbecause they provide structural features or biological activity that arenot required for the fusion molecules of the present invention. Thus,the term “Fc variant” comprises a molecule or sequence that lacks one ormore native Fc sites or residues that affect or are involved in (1)disulfide bond formation, (2) incompatibility with a selected host cell(3) N-terminal heterogeneity upon expression in a selected host cell,(4) glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

[0032] A “peptidomimetic” is a peptide analog that displays morefavorable pharmacological properties than their prototype nativepeptides, such as a) metabolic stability, b) good bioavailability, c)high receptor affinity and receptor selectivity, and d) minimal sideeffects. Designing peptidomimetics and methods of producing the same areknown in the art (see for example, U.S. Pat. Nos. 6,407,059 and6,420,118). Peptidomimetics may be derived from the binding site of theextracellular domain of IL-17, IL-17 receptor (IL-17R), IL-18, IL-18receptor (IL-18R), IL-18 binding protein (IL-18BP), CD30, CD30 ligand(CD30-L), 4-1BB, 4-1BB ligand (4-1BB-L), OX40, OX40 ligand (OX40-L)and/or CD39. In alternative embodiments, a peptidomimetic comprisesnon-peptide compounds having the same three-dimensional structure aspeptides derived from IL-17, IL-17 receptor (IL-17R), IL-18, IL-18receptor (IL-18R), IL-18 binding protein (IL-18BP), CD30, CD30 ligand(CD30-L), 4-1BB, 4-1BB ligand (4-1BB-L), OX40, OX40 ligand (OX40-L)and/or CD39, or compounds in which part of a peptide from the moleculeslisted above is replaced by a non-peptide moiety having the samethree-dimensional structure.

[0033] A “mimotope” is defined herein as peptide sequences that mimicbinding sites on proteins (see, Partidos, CD, et al., Combinatorial Chem& High Throughput Screening, 2002 5:15-27). A mimotope may have thecapacity to mimic a conformationally-dependent binding site of aprotein. The sequences of these mimotopes do not identify a continuouslinear native sequence or necessarily occur in a naturally-occurringprotein. Mimotpes and methods of production are taught in U.S. Pat. No.5,877,155 and U.S. Pat. No. 5,998,577, which are incorporated byreference in their entireties.

[0034] The term “acidic residue” refers to amino acid residues in D- orL-form having sidechains comprising acidic groups. Exemplary acidicresidues include D and E.

[0035] The term “amide residue” refers to amino acids in D- or L-formhaving sidechains comprising amide derivatives of acidic groups.Exemplary residues include N and Q.

[0036] The term “aromatic residue” refers to amino acid residues in D-or L-form having sidechains comprising aromatic groups. Exemplaryaromatic residues include F, Y, and W.

[0037] The term “basic residue” refers to amino acid residues in D- orL-form having sidechains comprising basic groups. Exemplary basicresidues include H, K, and R.

[0038] The term “hydrophilic residue” refers to amino acid residues inD- or L-form having sidechains comprising polar groups. Exemplaryhydrophilic residues include C, S, T, N, and Q.

[0039] The term “nonfunctional residue” refers to amino acid residues inD- or L-form having sidechains that lack acidic, basic, or aromaticgroups. Exemplary nonfunctional amino acid residues include M, G, A, V,I, L and norleucine (Nle).

[0040] The term “neutral hydrophobic residue” refers to amino acidresidues in D- or L-form having sidechains that lack basic, acidic, orpolar groups. Exemplary neutral hydrophobic amino acid residues includeA, V, L, I, P, W, M, and F.

[0041] The term “polar hydrophobic residue” refers to amino acidresidues in D- or L-form having sidechains comprising polar groups.Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C,Q, and N.

[0042] The term “hydrophobic residue” refers to amino acid residues inD- or L-form having sidechains that lack basic or acidic groups.Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M,F, T, G, S, Y, C, Q, and N.

[0043] The term “subject” as used herein, refers to mammals. Forexample, mammals contemplated by the present invention include humans;primates; pets of all sorts, such as dogs, cats, etc.; domesticatedanimals, such as, sheep, cattle, goats, pigs, horses and the like;common laboratory animals, such as mice, rats, rabbits, guinea pigs,etc.; as well as captive animals, such as in a zoo or free wild animals.Throughout the specification, the term host is used interchangeably withsubject.

[0044] As used herein the singular forms “a”,“and”,and “the” includeplural referents unless the context clearly dictates otherwise. Thus,for example, reference to “an immunization” includes a plurality of suchimmunizations and reference to “the cell” includes reference to one ormore cells and equivalents thereof known to those skilled in the art,and so forth. All technical and scientific terms used herein have thesame meaning as commonly understood to one of ordinary skill in the artto which this invention belongs unless clearly indicated otherwise.

[0045] It is understood that the various embodiments of this inventionare not limited to the particular methodology, protocols, cell lines,animal species or genera, constructs, and reagents described, as suchmay vary. It is also understood that the terminology used herein is forthe purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

I. IL-17, IL-18, 4-1BB, CD30 and OX40 Antagonists

[0046] A. IL-17 Antagonists

[0047] Embodiments of the present invention provide compositions andmethods for the treatment of cardiovascular disease comprising IL-17antagonists. Studies presented herein demonstrate that IL-17 and IL-18are elevated in human patients having various forms and severity ofcardiovascular disease. These studies demonstrate that circulatinglevels of IL-17 and/or IL-18 correlate with severity of cardiovasculardisease. Furthermore, plasma levels of IL-17 and/or IL-18 are elevatedin a cardiac myosin-induced myocarditis model and correlate with diseaseseverity. Therefore, IL-17 and/or IL-18 are implicated in cardiovasculardisease and provide a rational basis for treating cardiovascular diseaseby administering IL-17 and/or IL-18 antagonists, alone or incombination. In addition, IL-17 and/or IL-18 are prognostic indicatorsof cardiovascular disease and disease severity. IL-17 and/or IL-18 arealso prognostic indicators of donor adequacy and post-transplantoutcome. Therefore, further embodiments of the invention include assaysfor measuring IL-17 and/or IL-18 levels in subjects being screened forcardiovascular disease, cardiovascular disease severity, donor adequacyand post-transplant outcome.

[0048] An IL-17 antagonist is defined herein as an entitiy that iscapable of reducing the effective amount of endogenous IL-17 in asubject, by either partially or completely blocking the interaction ofIL-17 and the IL-17 receptor and thereby inhibiting IL-17-mediatedsignaling via membrane-bound IL-17 receptor, as well as partially orcompletely inhibiting the subsequent biological effects of activatingthe IL-17 receptor. An IL-17 antagonist may bind to IL-17 or to theIL-17 receptor.

[0049] Such IL-17 antagonists include, but are not limited to: solubleforms of IL-17 receptor; antibodies directed against IL-17 thatspecifically bind IL-17 and partially or completely inhibit binding ofIL-17 to IL-17 receptor; antibodies, fusion proteins and/or peptibodiesdirected against IL-17 receptor that specifically bind IL-17 receptorand inhibit binding of IL-17 without themselves activating the IL-17receptor; molecules that bind IL-17 or IL-17 receptor and inhibit theinteraction thereof, such as IL-17 and/or IL-17 receptor peptidomimeticsand/or mimotopes. As used herein, when reference is made to making IL-17antagonists based on IL-17 or IL-17 receptor, it is understood that theterms IL-17 and IL-17 receptor also encompass fragments, variants,muteins, derivatives and fusion proteins thereof, as described in detailbelow.

[0050] Biological activity of IL-17 and IL-17R is defined herein ascomprising binding of IL-17 to the IL-17R and activation of the IL-17R;proinflammatory effects; increased production of cytokines andchemokines, such as but not limited to, IL-6, IL-8, G-CSF, GM-CSF,MCP-1, Groα, PGE2, as well as induction of costimulatory molecule ICAM.The IL-17:IL-17R interaction also has the biological activities ofrecruiting monocytes and neutrophils, up-regulation of iNOS, NO andCOX-2; activation of all three subgroups of MAPKs (the p44 and p42extracellular signal-regulated kinases ERK1 and ERK2), NFκB,stress-induced Jun NH2-terminal kinases (JNK1 and JNK2) and p38. Ofcourse, it is understood that intermediate pathways that culminate insuch biological activities are also included in the definition ofbiological activity for IL-17 and IL-17R.

[0051] IL-17 antagonists may comprise or be developed from IL-17receptor polypeptide and/or polynucleotide sequences, as well asfragments, variants, muteins, derivatives and fusion proteins thereof.The isolation, cloning, preparation and characterization of human IL-17receptor (referred to interchangably as IL-17R or huIL-17R) aredescribed in U.S. Pat. No. 5,869,286 and U.S. Pat. No. 6,072,033, whichare incorported herein by reference in their entirety. The full-lengthcDNA sequence for human IL-17R is provided in SEQ ID NO:3 and thecorresponding amino acid sequence is provided in SEQ ID NO:4.

[0052] The human IL-17 receptor has an N-terminal signal peptide with apredicted cleavage site between amino acid 27 and 28. The signal peptideis followed by a 293 amino acid extracellular domain, a 21 amino acidtransmembrane domain, and a 525 amino acid cytoplasmic tail. Solublepolypeptides are polypeptides that are capable of being secreted fromthe cells in which they are expressed. A secreted soluble polypeptidemay be identified (and distinguished from its non-soluble membrane-boundcounterparts) by separating intact cells which express the desiredpolypeptide from the culture medium, e.g., by centrifugation, andassaying the medium (supernatant) for the presence of the desiredpolypeptide. The presence of the desired polypeptide in the mediumindicates that the polypeptide was secreted from the cells and thus is asoluble form of the polypeptide. The use of soluble forms of IL-17receptor is advantageous for many applications. Purification of thepolypeptides from recombinant host cells is facilitated, since thesoluble polypeptides are secreted from the cells. Moreover, solublepolypeptides are generally more suitable than membrane-bound forms forparenteral administration and for many enzymatic procedures. Solubleforms of IL-17R that are useful in the methods of treatingcardiovascular disease include the extracellular domain (residues 1-320of SEQ ID NO:4 or residues 28-320 which excludes the signal peptide) ora fragment of the extracellular domain that has the properties ofantagonizing or preventing binding of IL-17 to endogenous IL-17R.Soluble IL-17R also includes those polypeptides which include part ofthe transmembrane region, provided that the soluble IL-17R is capable ofbeing secreted from a cell, and preferably retains the capacity to bindIL-17 and effectuate its biological effects.

[0053] Other forms of the IL-17R that are useful in the presentinvention include muteins and variants (also referred to as analogs),such as naturally occurring variants, that are substantially homologousto the native IL-17R of SEQ ID NO:4 and as described in U.S. Pat. No.6,072,033 that retain biological activity of IL-17R.

[0054] The invention further encompasses IL-17 antagonists derived fromIL-17R polynucleotide sequences. Embodiments of the invention includefull length nucleic acid molecules encoding soluble IL-17R as well asisolated fragments and oligonucleotides derived from the nucleotidesequence of SEQ ID NO:3. Such nucleic acid sequences may includenucleotides 178-1494 of SEQ ID NO:3 or a fragment thereof, and DNAand/or RNA sequences that hybridize to the coding region of thenucleotide sequence of SEQ ID NO:3, or its complement, under conditionsof moderate stringency, and which encode polypeptides or fragmentsthereof of the invention.

[0055] In other embodiments, IL-17 antagonists may comprise or bedeveloped from IL-17 polynucleotide and/or polypeptide sequences. Thefull-length cDNA sequence for human IL-17 is provided in SEQ ID NO: 1and the corresponding amino acid sequence is provided in SEQ ID NO: 2.Commercially available recombinant human IL-17 is also available, forexample, from R&D Systems, Minneapolis, MN. IL-17 polypeptides, as wellas biologically active fragments or derivatives thereof, may be used togenerate antibodies that specifically bind to IL-17 and have thecapacity of partially or completely blocking IL-17 binding to the IL-17receptor.

[0056] In further embodiments, IL-17 antagonists are small molecules andpolypeptide mimetics, such as but not limited to, peptidomimetics,peptibodies and/or mimotopes developed from the polypeptide sequence ofIL-17 (SEQ ID NO:2) and/or IL-17R (SEQ ID NO:4). Polypeptide mimeticsare peptide-containing molecules which mimic elements of proteinsecondary structure. Polypeptide mimetics, such as peptidomimetics andmimotopes, may be developed through techniques known in the art, such ascombinatorial peptide libraries.

[0057] An IL-17 polypeptide mimetic based on the amino acid sequence ofIL-17 will bind to IL-17R without activating the IL-17R and stericallyhinder binding of endogenous IL-17. Conversely, an IL-17R polypeptidemimetic based on the amino acid sequence of IL-17R will bind to IL-17and sterically hinder IL-17 from binding to endogenous IL-17R. IL-17peptide mimetics may be used to antagonize IL-17 in a subject andthereby reduce the proinflammatory effects of IL-17. As such, IL-17polypeptide mimetics may be used to treat inflammatory and/orimmunoregulatory processes associated with cardiovascular disease.

[0058] Other forms of the IL-17 that are useful in the present inventioninclude muteins and variants (also referred to as analogs), such asnaturally occurring variants, that are substantially homologous to thenative IL-17 of SEQ ID NO:2 that retain biological activity of IL-17.For example, IL-17 homologues B, C, D, E and F. This inventionadditionally provides for the use of IL-17 antagonists in themanufacture of a medicament for the treatment of cardiovascular disease.This invention further provides for the use of polynucleotides encodingIL-17 antagonists in the manufacture of a medicament for the treatmentof cardiovascular disease.

[0059] B. IL-18 Antagonists

[0060] Embodiments of the present invention provide compositions andmethods for the treatment of cardiovascular disease comprising IL-18antagonists. An IL-18 antagonist is defined herein as an entitiy that iscapable of reducing the effective amount of endogenous IL-1 8, by eitherpartially or completely blocking the interaction of IL-18 and the IL-18Rand thereby inhibiting IL-18-mediated signaling via membrane-boundIL-18R, as well as partially or completely inhibiting the subsequentbiological effects of activating the IL-18 receptor. An IL-18 antagonistmay bind to IL-18 or to the IL-18R. Antagonists derived from the IL-18Rand IL-18 Binding Protein (e.g. soluble forms that bind IL-18) competefor IL-18 with IL-18R on the cell surface, thus inhibiting IL-18 frombinding to cells, thereby preventing IL-18 from manifesting itsbiological activities.

[0061] IL-18: IL-18R biological activity, is defined herein asincluding, but is not limted to, binding of IL-18 to the IL-18R andactivation of the IL-18R; regulation of innate and acquired immuneresponses; proinflammatory effects; induction of T-lymphocyte helpercell type 1 responses (Th1); enhance cell-mediated cytotoxicity; IFN-γinduction; enhanced production of GM-CSF and IL-2; potentiation ofanti-CD3 induced T-cell proliferation; increased Fas-mediated kiling bynatural killer cells (NK cells) and CD4+ Th1 cells; increased apoptoticdeath via the Fas-FasL pathway; up-regulation of FasL expression;induction of T-lymphocyte helper cell type 2 responses (Th2) in T-cellsand NK cells; stimulation of basophils and mast cells to produce Th2cytokines and histamine; induction of IgE production

[0062] Such IL-18 antagonists include, but are not limited to: solubleforms of IL-18R; IL-18 Binding Protein; antibodies directed againstIL-18 that specifically bind IL-18 and partially or completely inhibitbinding of IL-18 to IL-18R; antibodies, fusion proteins and/orpeptobodies directed against IL-18R that specifically bind IL-18R andinhibit receptor binding of IL-18 without themselves transducing asignal via IL-18R; small molecules that bind IL-18 or IL-18R thatinhibit the interaction thereof, such as IL-18 and/or IL-18Rpeptidomimetics and/or mimotopes. As used herein, when reference is madeto making IL-18 antagonists based on IL-18, IL-18 Binding Protein orIL-18 receptor, it is understood that the terms IL-18, IL-18 BindingProtein and IL-18 receptor also encompass fragments, variants, muteins,derivatives and fusion proteins thereof, as described in detail below.

[0063] The isolation, cloning, preparation and characterization of humanIL-18 receptor (referred to interchangably as IL-18R or huIL-18R) aredescribed in U.S. Pat. No. 6,087,116 and U.S. patent application Ser.No. 09/621,502 (PCT Publication WO 99/37772), which are incorportedherein by reference in their entirety. The IL-18 receptor is aheterodimeric protein containing an IL-18 binding subunit termedIL-1Rrp1, and an accessory subunit termed AcPL. Although the IL-Rrp1subunit alone will bind IL-18, its affinity for IL-18 is increaseddramatically when present in a heterodimeric complex with the AcPLsubunit.

[0064] The IL-1Rrp1 polynucleotide sequence and corresponding amino acidsequence that it encodes are provided as SEQ ID NO:5 and SEQ ID NO:6,respectively. The soluble extracellular portion of the IL-1Rrp1 subunitthat binds IL-18 is represented by amino acids 20 to 329 of SEQ ID NO:6;cleavage of the signal sequence occurs just after amino acid residue 19of SEQ ID NO:6. However, fragments as small as amino acid residues 20 to123 and amino acid residues 20 to 226 of SEQ ID NO:6 have been reportedto bind IL-18 and can also be used. The IL-1Rrp1 polypeptide is alsodescribed in U.S. Pat. No. 5,776,731, incorporated in its entirety byreference herein.

[0065] The AcPL polynucleotide sequence and the amino acid sequence thatit encodes are provided herein as SEQ ID NO:7 and SEQ ID NO:8,respectively. The mature extracellular domain of AcPL consists of aminoacids 15 to 356 of SEQ ID NO:8; cleavage of the signal sequence occursjust after amino acid residue 14 of SEQ ID NO:8. The AcPL polypeptide,and soluble extracellular fragments thereof, are also described in inU.S. patent application Ser. No. 09/621,502 (PCT Publication WO99/37772), incorporated herein by reference in its entirety.

[0066] One embodiment of a soluble form of an IL-18 receptor for use inthe methods of the present invention comprises amino acids 1-329sequence of SEQ ID NO:6; alternative embodiments of a soluble form of anIL-18 receptor comprises amino acids 20-329 after cleavage of the signalsequence of SEQ ID NO:6. A further embodiment of a soluble form of IL-18receptor is a heterodimeric receptor that includes at least amino acidresidues 20-123, 20-226 or 20-329 of SEQ ID NO:6 (the IL-1Rrp1 subunit),and at least amino acids 15-340 of SEQ ID NO:8 (the AcPL subunit), in acovalent or non-covalent association.

[0067] Additional IL-18 antagonists comprise the IL-18 Binding Protein.PCT Publication WO 99/09063 describes the IL-18 binding protein,including useful soluble fragments thereof. One embodiment of a humanIL-18 Binding Protein is the “a” isoform having the polynucleotidesequence of SEQ ID NO:9 and the corresponding amino acid sequence of SEQID NO:10. Of course, other IL-18 Binding Protein isoforms that areantagonistic to IL-18 binding to IL-18R may be used, such as the b, cand d isoforms. The polynucleotide and amino acid sequences for the b, cand d isoforms are known in the art and readily available (see forexample, Kim, S.-H., et al., PNAS 97:3 1190-1195 (2000)). A particularlyuseful form of the IL-18 binding protein is a fusion with an Fc domainof an antibody. The amino acid sequence of an example of such a fusionprotein, termed IL-18BP-Fc herein, is presented in SEQ ID NO:11. This422 amino acid protein, when expressed in a mammalian cell, will besecreted; the mature secreted form of the protein contains amino acidresidues 29-422. Of these residues, amino acid residues 29-192 representthe IL-18 binding protein portion of the molecule, and amino acidresidues 193-422 represent the Fc portion of the molecule. The Fc regionfacilitates purification and dimerization of the fusion polypeptide.

[0068] IL-18 antagonists may also comprise or be developed from IL-18polynucleotide and/or polypeptide sequences. Human IL-18 has beenrecombinantly produced from a cloned cDNA, as described in U.S. Pat. No.5,891,663 and cloned genomic DNA, as disclosed in U.S. Pat. No.6,060,283, which are incorporated by reference in their entirety. Thefull-length cDNA sequence in provided in SEQ ID NO:12 with thecorresponding amino acid sequence in SEQ ID NO:13. The amino acidsequence for ICE-processed human IL-18 provided in SEQ ID NO:14.Commercially available recombinant human IL-18 is available, forexample, from R&D Systems, Minneapolis, MN. IL-18 polypeptides as wellas biologically active fragments or derivatives thereof may be used togenerate antibodies that specifically bind to IL-18 and have thecapacity of partially or completely blocking IL-18 binding to the IL-18receptor.

[0069] In one embodiment, IL-18 antagonists are polypeptide mimetics,such as, but not limited to peptidomimetics, peptibodies and/ormimotopes developed from the polypeptide sequence of IL-18 (SEQ ID NO:13and/or 14). Polypeptide mimetics may be developed through techniquesknown in the art, such as combinatorial peptide libraries. Polypeptidemimetics are peptide-containing molecules which mimic elements ofprotein secondary structure. An IL-18 polypeptide mimetic based on theamino acid sequence of IL-18 will bind to IL-18 receptor withoutactivating the IL-18 receptor and sterically hinder binding ofendogenous IL-18. IL-18 peptide mimetics may be used to antagonize IL-18in a subject and thereby reduce the proinflammatory effects of IL-18. Assuch, IL-18 polypeptide mimetics may be used to treat inflammatoryand/or immunoregulatory processes associated with cardiovasculardisease.

[0070] Other embodiments of IL-18, IL-18 receptor and IL-18 BindingProtein that may be used as IL-18 antagonists include muteins andvariants (as described in greater detail below), such as naturallyoccurring variants, that are substantially homologous to the nativeIL-18 of SEQ ID NO:13 and/or 14, IL-18 receptor of SEQ ID NOs:6 and 8,and IL-18 Binding Protein of SEQ ID NO:10 that retain biologicalactivity. Biological activity, in this instance, is the capacity to bindits cognate partner.

[0071] This invention additionally provides for the use of IL-18antagonists in the manufacture of a medicament for the treatment ofcardiovascular disease. This invention further provides for the use ofpolynucleotides encoding IL-18 antagonists in the manufacture of amedicament for the treatment of cardiovascular disease.

[0072] C. 4-1BB Antagonists

[0073] Further embodiments of the present invention provide compositionsand methods for the treatment of cardiovascular disease comprising 4-1BBantagonists.

[0074] Examples 7 and 8 describe studies demonstrating a role for the4-1BB/4-1BBL immune co-stimulatory pathway in Adriamycin®-inducedcardiomyopathy, as well as deomonstrating a novel cardiac expressionpattern of 4-1BB and implicating apoptosis as a mechanism ofco-stimulatory contribution to Adriamycin®-induced cardiomyopathy. Morespecifically, 4-1BBL deficient mice and 4-1BBL decoy receptor-treatedmice conferred partial resistance to adriamycin induced cardiac damage,whereas 4-1BB activating antibody accelerated onset of damage, implyingthe contribution of 4-1BB to Adriamycin® effects in heart. Apoptosis,measured by TUNEL, sub-G1 DNA and activated caspase-3, was increased inAdriamycin®-treated wild type myocardium, but reduced in 4-1BBL−/−.Phosphorylation of Akt was selectively suppressed by Adriamycin®, butmaintained by loss of 4-1BBL, indicating the modulation of apoptosis byco-stimulatory pathway in heart is possibly through Akt, but not Jnk andp38 signaling. The consistency of decreased index of apoptosis and theimproved cardiac function in 4-1BBL−/− suggests apoptosis play a pivotalrole in Adriamycin®-induced cardiac deficiency.

[0075] A single retroorbital (RO) injection of adriamycin (22.5 mg/kg)leads to progressive cardiac dysfunction without evidence ofinflammatory infiltration. In this model of non-inflammatory,drug-induced cardiomyopathy, 4-1BBL−/− mice have substantially improvedcardiac function by echocardiography. Furthermore, m4-1BB Fc (a solubledecoy receptor for 4-1BBL) reduced ADR cardiac dysfunction, while anagonistic antibody to 4-1BB (M6) accelerated and exacerbated cardiacdysfunction. While no inflammatory infiltrate is observed in thisAdr-cardiomyopathy, we found expression of 4-1BB induced on 1-5% ofcardiac interstitial cells within 2 days after Adr. Cardiac apoptosis,measured by TUNEL and sub-G1 DNA, is increased 3 days after ADR(45mg/kg), concomitant with the increased expression of 4-1BB oninterstitial cells. Chronic ongoing apoptosis, determined 5 weeks afterAdr challenge when cardiac dysfunction is maximal in wild type butlargely absent in 4-1BBL−/− mice, was lower in 4-1BBL−/− mice (1.5-foldvs baseline), compared to WT mice (4 fold). In a separate study, caspase3 activation, determined by Western blot, was increased at 48 to 72 hrspost-ADR (45 mg/kg). In contrast, ADR did not induce caspase 3 cleavagein 4-1BBL−/− myocardium. Determined by western blot, adriamycin reducedphosphorylation of Akt in wild type but not 4-1BBL−/− hearts.Phosphorylation of JNK and p38 was not impacted by Adr. In summary,4-1BB/4-1BBL immune co-stimulatory pathway contributes to ADR-inducedcardiomyopathy, possibly, through modulation of Akt signaling toregulate apoptosis in the heart.

[0076] A 4-1BB antagonist is defined herein as an entitiy that iscapable of reducing the effective amount of available endogenous 4-1BBand/or 4-1BB ligand (4-1BB-L), by either partially or completelyblocking the interaction of 4-1BB and 4-1BB-L and thereby inhibiting4-1BB-mediated signaling via 4-1BB-L and 4-1BB-L-mediated signaling via4-1BB, as well as the subsequent biological effects of activating 4-1BBand/or 4-1BB-L. In other words, because the 4-1BB:4-1BB-L interactionexhibits bi-directional signalling, a 4-1BB antagonist may bind either4-1BB or 4-1BB-L so long as the antagonist does not itself activate4-1BB or 4-1BB-L. Such 4-1BB antagonists include, but are not limitedto: soluble forms of 4-1BB; antibodies, fusion proteins and/orpeptibodies directed against 4-1BB that specifically bind 4-1BB andpartially or completely inhibit binding of 4-1BB to 4-1BB-L; antibodies,fusion proteins and/or peptibodies directed against 4-1BB thatspecifically bind 4-1BB and inhibit binding of 4-1BB-L withoutthemselves transducing a signal via 4-1BB; molecules that bind 4-1BB or4-1BB-L and inhibit the interaction thereof, such as 4-1BB and/or4-1BB-L small molecules, peptidomimetics and/or mimotopes, and/orpolypeptides comprising all or portions of 4-1BB or 4-1BB-L or modifiedvariants thereof, including genetically-modified muteins, multimericforms and sustained-release formulations thereof.

[0077] 4-1BB:4-1BB-L biological activity, is defined herein asincluding, but is not limted to, binding of 4-1BB-L to 4-1BB andactivation of one or both of 4-1BB and 4-1BB-L; costimulatory activityon T lymphocytes; activation and differentiation of CD4+and CD8+ cells;signal transduction through TRAF pathways (TRAF1, TRAF2 and TRAF3) andactivation of NFκB and AP-1; inhibition of activation-induced celldeath; facilitation of B-cell proliferation and monocyte activation;up-regulation of cytokines including, but not limited to, IL-6, IL-8 andTNF-α; up-regulation of adhesion molecules, such as ICAM;down-regulation of FcγRIII; production of M-CSF in monocytes; monocyteproliferation; and inhibition of T-cell proliferation induced byanti-CD3 antibodies.

[0078] 4-1BB antagonists may comprise or be developed from 4-1BB-Lpolypeptide and polynucleotide sequences. The isolation, cloning,preparation and characterization of human 4-1BB-L (referred tointerchangably as hu4-1BB-L) is described in U.S. Pat. No. 5,674,704,which is incorported herein by reference in its entirety. 4-1BB-L refersto a genus of mammalian polypeptides that are capable of binding 4-1BB.4-1BB-L is a type II extracellular membrane polypeptide with anintracellular (cytoplasmic) domain at the N-terminus of the polypeptide(amino acids 1-25 of SEQ ID NO:15), followed by a transmembrane regionpolypeptide (amino acids 26-48 of SEQ ID NO:15), and an extracellular(receptor-binding) domain at the C-terminus of the polypeptidepolypeptide (amino acids 49-254 of SEQ ID NO:15). Soluble 4-1BB-Lpolypeptides may be derived from the extracellular domain, as describedbelow. The full-length cDNA sequence for human 4-1BB-L is provided inSEQ ID NO:14 and the corresponding amino acid sequence is provided inSEQ ID NO:15. The human 4-1BB-L protein comprises a cytoplasmic domain(amino acids 1-25), a transmembrane region (amino acids 26-48), and anextracellular domain (amino acids 49-254 of SEQ ID NO:15).

[0079] In addition, 4-1BB antagonists may comprise or be developed from4-1BB polypeptide and polynucleotide sequences. The polynucleotidesequence of a human 4-1BB cDNA and the amino acid sequence encodedthereby are presented in SEQ ID NO:17 and SEQ ID NO:18, respectively.The human 4-1BB protein comprises an N-terminal signal sequence (aminoacids −23 to −1 of SEQ ID NO:18), an extracellular domain comprisingamino acids 1-163, a transmembrane region comprising amino acids164-190, and a cytoplasmic domain comprising amino acids 191-232.

[0080] Soluble forms of 4-1BB-L and 4-1BB proteins are provided herein.Soluble 4-1BB-L or 4-1BB polypeptides comprise all or part of theextracellular domain but lack the transmembrane region that would causeretention of the polypeptide on a cell membrane. Since the 4-1BB-Lprotein lacks a signal peptide, a heterologous signal peptide may befused to the N-terminus of soluble 4-1BB-L polypeptides to promotesecretion thereof. The signal peptide is cleaved from the protein uponsecretion from the host cell. Soluble 4-1BB-L polypeptides includefragments that retain the ability to bind 4-1BB, such as truncatedpolypeptides of the extracellular domain, and soluble 4-1BB polypeptidesinclude fragments that retain the ability to bind 4-1BB-L, such astruncated polypeptides of the extracellular domain 4-1BB. In alternativeembodiments, the soluble proteins may include part of the transmembraneregion or part of the cytoplasmic domain, provided that the protein iscapable of being secreted rather than retained on the cell surface.Examples of soluble polypeptides include those comprising the entireextracellular domain. Specific examples include, but are not limited toa soluble human 4-1BB-L polypeptide comprising amino acids 49-254 of SEQID NO:16 and a soluble human 4-1BB polypeptide comprising amino acids1-163 of SEQ ID NO:18.

[0081] In one embodiment, 4-1BB antagonists are polypeptide mimetics,such as but not limited to, peptidomimetics, peptibodies and/ormimotopes developed from the polypeptide sequence of 4-1BB-L (SEQ IDNO:16) and 4-1BB (SEQ ID NO:18). Polypeptide mimetics may be developedthrough techniques known in the art, such as combinatorial peptidelibraries. Polypeptide mimetics are peptide-containing molecules whichmimic elements of protein secondary structure. A 4-1BB polypeptidemimetic based on the amino acid sequence of 4-1BB will bind to 4-1BB-Lwithout activating 4-1BB-L and sterically hinder binding of endogenous4-1BB. Similarly, a 4-1BB-L polypeptide mimetic based on the amino acidsequence of 4-1BB-L will bind to 4-1BB without activating 4-1BB andsterically hinder binding of endogenous 4-1BB-L. 4-1BB and 4-1BB-Lpeptide mimetics may be used to antagonize their respective cognates ina subject and thereby reduce the proinflammatory effects of the4-1BB/4-1BB-L interaction. As such, 4-1BB antagonists in the form ofpolypeptide mimetics may be used to treat inflammatory and/orimmunoregulatory processes associated with cardiovascular disease.

[0082] Other embodiments of 4-1BB and 4-1BB-L that may be used as 4-1BBantagonists include muteins and variants (as described in greater detailbelow), such as naturally occurring variants, that are substantiallyhomologous to the native 4-1BB-L (SEQ ID NO:16) and 4-1BB (SEQ ID NO:18)polypeptide sequences that retain biological activity. Biologicalactivity, in this instance, is the capacity to bind its cognate partner.

[0083] This invention additionally provides for the use of 4-1BBantagonists in the manufacture of a medicament for the treatment ofcardiovascular disease. This invention further provides for the use ofpolynucleotides encoding 4-1BB antagonists in the manufacture of amedicament for the treatment of cardiovascular disease.

[0084] D. CD30 Antagonists

[0085] Further embodiments of the present invention provide compositionsand methods for the treatment of cardiovascular disease comprising oneor more CD30 antagonists. A CD30 antagonist is defined herein as anentity that is capable of reducing the effective amount of endogenousCD30 ligand (CD30-L), by either partially or completely blocking theinteraction of CD30-L and CD30 and thereby inhibiting CD30-mediatedsignaling via membrane-bound CD30, as well as partially or completelyinhibiting the subsequent biological activity of activating CD30. A CD30antagonist may bind to either CD30-L or CD30.

[0086] The biological activity of CD30: CD30-L includes, but is notlimited to, binding of CD30-L to CD30 and activation of CD30;intracellular activation of NF-κB, cytokine release and/or proliferationof CD30+ cells; proliferation of T-cells in the presence of an anti-CD3co-stimulus.

[0087] Such CD30 antagonists include, but are not limited to: solubleforms of CD30-L and CD30; fragments of CD30-L that bind CD30 and inhibitbinding of CD30-L without activating membrane-bound CD30; fragments ofCD30 that bind CD30-L and inhibit binding of CD30-L to CD30; antibodies,fusion proteins and/or peptibodies directed against CD30-L thatspecifically bind CD30-L and partially or completely inhibit binding ofCD30-L to CD30; antibodies, fusion proteins and/or peptibodies directedagainst CD30 that specifically bind CD30 and inhibit binding of CD30-Lwithout themselves activating the CD30; small molecules that bind CD30-Lor CD30 and inhibit the interaction thereof, such as CD30-L and/or CD30peptidomimetics and/or mimotopes. As used herein, when reference is madeto CD30 antagonists based on CD30-L or CD30, it is understood that theterms CD30-L and CD30 also encompass fragments, variants, muteins,derivatives and fusion proteins thereof, as described in detail below.

[0088] CD30 antagonists may comprise or be developed from CD30-Lpolynucleotide and polypeptide sequences. The isolation, cloning,preparation and characterization of human CD30-L is described in U.S.Pat. No. 5,480,981, which is incorported herein by reference in itsentirety. As mentioned above, embodiments of the present inventioninclude anti-CD30-L antibodies as CD30 antagonists. Examples ofantibodies that are directed against CD30-L that may be used to treatcardiovascular disease are described in U.S. Pat. No. 5,677,430, whichis incorporated by reference in its entirety.

[0089] The term “CD30-L” as used herein refers to a genus ofpolypeptides which are capable of binding CD30. As used herein, the term“CD30-L” includes membrane-bound proteins (comprising a cytoplasmicdomain, a transmembrane region, and an extracellular domain) as well astruncated proteins that retain the CD30-binding property. Such truncatedproteins include, for example, soluble CD30-L comprising only theextracellular (receptor binding) domain. CD30-L is expressed onmonocytes/macrophages, granulocytes, a subset of B cells and onactivated but not resting T cells. By binding with cell-surface CD30,CD30-L can induce murine B cell differentiation and can induce theproliferation of activated T cells in the presence of an anti-CD3co-stimulus (see, for example, Smith et al., Cell 73:1349-1360 (1993)).Moreover, CD30-L exhibits “reverse signaling,” that is, the cell surfaceCD30-L that is expressed on neutrophils and peripheral blood T cells canbe activated by cross-linking to stimulate metabolic activities in thosecells (Wiley et al., J Immunol 157: 3235-39 (1996)).

[0090] CD30-L proteins of the present invention include, but are notlimited to, human CD30-L comprising amino acids 1-215 of SEQ ID NO:20 or1-234 of SEQ ID NO:22; and proteins that comprise N-terminal,C-terminal, or internal truncations of the foregoing sequences, butretain the desired biological activity. Examples include human CD30-Lproteins comprising amino acids y to 234 of SEQ ID NO:22 wherein y is1-19 (i.e., the N-terminal amino acid is any one of amino acids 1-19 ofSEQ ID NO:22, and amino acid 234 is the C-terminal amino acid. Suchproteins, truncated at the N-terminus, are capable of binding CD30.

[0091] Alternative embodiments provide soluble CD30-L polypeptides.Soluble CD30-L polypeptides comprise all or part of the extracellulardomain of a native CD30-L but lack the transmembrane region that wouldcause retention of the polypeptide on a cell membrane. Since the CD30-Lprotein lacks a signal peptide, a heterologous signal peptide may befused to the N-terminus of a soluble CD30-L protein to promote secretionthereof. The signal peptide is cleaved from the CD30-L protein uponsecretion from the host cell. The soluble CD30-L polypeptides retain theability to bind the CD30 receptor. Soluble CD30-L may also include partof the transmembrane region or part of the cytoplasmic domain or othersequences, provided that the soluble CD30-L protein is capable of beingsecreted.

[0092] Examples of soluble CD30-L polypeptides include those comprisingthe entire extracellular domain of a native CD30-L protein or a fragmentof said extracellular domain that is capable of binding CD30. One suchsoluble CD30-L polypeptides comprise amino acids z to 215 (Asp) of thehuman CD30-L sequence of SEQ ID NO:20, wherein z is 44, 45, 46, or 47.In other words, the N-terminal amino acid of the soluble human CD30-L isselected from the amino acids in positions 44-47 of SEQ ID NO:20. DNAsequences encoding such soluble human CD30-L polypeptides include, butare not limited to, DNA sequences comprising a nucleotide sequenceselected from the group consisting of nucleotides 130-645, 133-645,136-645, and 139-645 of SEQ ID NO:19. Such sequences encode polypeptidescomprising amino acids 44-215, 45-215, 46-215, and 47-215, respectively,of SEQ ID NO:20. Production of one such soluble human CD30-L protein, inthe form of a fusion protein comprising amino acids 47-215 of SEQ IDNO:20 and an antibody Fc polypeptide, is illustrated in Example 11 ofU.S. Pat. No. 5,480,981.

[0093] CD30 antagonists may comprise or be developed from CD30polypeptide and/or polynucleotide sequences. Cloning and expression of agene encoding CD30 has been reported and CD30 has been characterized asa transmembrane protein that possesses substantial homology to the nervegrowth factor receptor superfamily (Durkop et al., Cell 1992, 68:421).The CD30 polynucleotide sequence reported in Durkop et al. supra ispresented in SEQ ID NO:23, and the amino acid sequence encoded therebyis presented in SEQ ID NO:24. The extracellular portion of human CD30corresponds to amino acids 1-390, or if the signal peptide is removed,to amino acids 19-390 of SEQ ID NO:24. The transmembrane regioncomprises amino acids 391-407 of SEQ ID NO:24. The phrase “solubleCD30“(sCD30) refers to soluble molecules that comprise all or part ofthe extracellular domain of the CD30 protein, and that retain thecapacity to bind specifically with CD30-L. The polynucleotide andpolypeptide sequences, as well as a description of how to make a CD30-Fcfusion protein, which may serve as a CD30 antagonist for the treatmentof cardiovascular disease, is described in detail in U.S. Pat. No.5,480,981, which is incorported herein by reference in its entirety.

[0094] In further embodiments, CD30 antagonists are polypeptidemimetics, such as, but not limited to peptidomimetics, peptibodiesand/or mimotopes developed from the polypeptide sequence of CD30-L (SEQID NOs:20 and/or 22) and/or CD30 (SEQ ID NO:24). Polypeptide mimeticsmay be developed through techniques known in the art, such ascombinatorial peptide libraries. Polypeptide mimetics arepeptide-containing molecules which mimic elements of protein secondarystructure. A CD30-L polypeptide mimetic based on the amino acid sequenceof CD30-L will bind to CD30 without activating CD30 and stericallyhinder binding of endogenous CD30-L. A CD30 receptor polypeptide mimeticbased on the amino acid sequence of CD30 receptor will bind to CD30-Land sterically hinder binding of endogenous CD30-L to CD30. CD30-L andCD30 peptide mimetics can be used to antagonize CD30-L binding to CD30in a subject and thereby reduce the proinflammatory effects of CD30-L.As such, CD30 antagonists in the form of polypeptide mimetics may beused to treat inflammatory and/or immunoregulatory processes associatedwith cardiovascular disease.

[0095] Other forms of CD30-L and CD30 that are useful in the presentinvention include muteins and variants (also referred to as analogs),such as naturally occurring variants, that are substantially homologousto the native CD30-L (SEQ ID NOs:20 and/or 22) or CD30 (SEQ ID NO:24)polypeptide sequences and as described in U.S. Pat. No. 5,480,981 thatretain biological activity.

[0096] This invention additionally provides for the use of CD30antagonists in the manufacture of a medicament for the treatment ofcardiovascular disease. This invention further provides for the use ofpolynucleotides encoding CD30 antagonists in the manufacture of amedicament for the treatment of cardiovascular disease.

[0097] E. OX40 Antagonists

[0098] Further embodiments of the present invention provide compositionsand methods for the treatment of cardiovascular disease comprising oneor more OX40 antagonists. A OX40 antagonist is defined herein as anentity that is capable of reducing the effective amount of endogenousOX40 ligand (OX40-L), by either partially or completely blocking theinteraction of OX40-L and OX40 and thereby inhibiting OX40-mediatedsignaling via membrane-bound OX40, as well as partially or completelyinhibiting the subsequent biological activity of activating OX40. A OX40antagonist may bind to either OX40-L or OX40.

[0099] OX40: OX40-L biological activity, is defined herein as including,but is not limted to, binding of OX40-L to OX40 and activation of OX40;costimulatory activity on T lymphocytes; cytokine production, includingIL-4; promoting the survival and proliferation of CD4+ T cells;prolongation of immune responses; enhancing effector and memory-effectorT cell fucntion by upregulating IL-2 production and increasing thelife-span of effector T cells; and, enhanced tumor-specific immunity.

[0100] Such OX40 antagonists include, but are not limited to: solubleforms of OX40-L and OX40; fragments of OX40-L that bind OX40 and inhibitbinding of OX40-L without activating membrane-bound OX40; fragments ofOX40 that bind OX40-L and inhibit binding of OX40-L to OX40; antibodies,fusion proteins and/or peptibodies directed against OX40-L thatspecifically bind OX40-L and partially or completely inhibit binding ofOX40-L to OX40; antibodies, fusion proteins and/or peptibodies directedagainst OX40 that specifically bind OX40 and inhibit binding of OX40-Lwithout themselves activating the OX40; small molecules that bind OX40-Lor OX40 and inhibit the interaction thereof, such as OX40-L and/or OX40peptidomimetics and/or mimotopes. As used herein, when reference is madeto OX40 antagonists based on OX40-L or OX40, it is understood that theterms OX40-L and OX40 also encompass fragments, variants, muteins,derivatives and fusion proteins thereof, as described in detail below.

[0101] OX40 antagonists may comprise or be developed from OX40polynucleotide and polypeptide sequences. The OX-40 receptor, alsoreferred to as OX40, ACT-4 and ACT35, is a protein expressed on thesurface of antigen-activated mammalian CD4+ T-cells. DNA sequencesencoding mouse, rat and human OX-40 receptor homologs have been clonedand sequenced (see, Mallet, et al., EMBO, 9:1063-1068 (1990);Calderhead, et al., J Immunol, 151:5261-5271 (1993); Latza, et al., Eur.J. Immunol. 24:677-683 (1994); and WO 95/12673). The isolation, cloning,and characterization of human OX40 is described in U.S. Pat. Nos.5,821,332 and 6,277,962 B1, which are incorported herein by reference intheir entirety. As mentioned above, embodiments of the present inventioninclude anti-OX40 antibodies as OX40 antagonists. Examples of antibodiesthat are directed against OX40 are described in U.S. Pat. Nos. 5,821,332and 6,277,962 B1.

[0102] OX40 proteins of the present invention include, but are notlimited to, human OX40 comprising amino acids 1-277 of SEQ ID NO:28; andproteins that comprise N-terminal, C-terminal, or internal truncationsof the foregoing sequences, but retain the desired biological activity.DNA sequences encoding such human OX40 polypeptides include, but are notlimited to, DNA sequences comprising the nucleotide sequence of SEQ IDNO:27.

[0103] Alternative embodiments provide soluble OX40 polypeptides.Soluble OX40 polypeptides comprise all or part of the extracellulardomain of a native OX40 but lack the transmembrane region that wouldcause retention of the polypeptide on a cell membrane. The soluble OX40polypeptides retain the ability to bind the OX40-L. Soluble OX40polypeptides may also include part of the transmembrane region or partof the cytoplasmic domain or other sequences, provided that the solubleOX40 protein is capable of being secreted. The putative signal sequenceis from amino acids 1-22 or 1-24 and the extracellular domain spanningamino acids 23-212 or 24-212 or 24-212 and the transmembrane sequencespanning amino acids 213-240 of SEQ ID NO:28.

[0104] Examples of soluble OX40 polypeptides include those comprisingthe entire extracellular domain of a native OX40 protein or a fragmentof said extracellular domain that is capable of binding OX40-L. One suchsoluble OX40-L polypeptides comprise amino acids z to 213 SEQ ID NO:28,wherein z is 22, 23, 24, or 25. In other words, the N-terminal aminoacid of the soluble human OX40-L is selected from the amino acids inpositions 22-25 of SEQ ID NO:28.

[0105] OX40 antagonists may comprise or be developed from OX40-Lpolynucleotide and polypeptide sequences. The isolation, cloning,preparation and characterization of human OX40-L is described in U.S.Pat. Nos. 6,156,878 and 6,242,566 B1, as well as U.S. application Ser.Nos: US 2001/0044523 A1 and US 2002/0077460 A1, which are incorportedherein by reference in their entirety. As mentioned above, embodimentsof the present invention include anti-OX40-L antibodies as OX40antagonists. Examples of antibodies that are directed against OX40-L aredescribed in U.S. Pat. No. 6,156,878 and 6,242,566 B1, as well as U.S.application Ser. Nos: US 2001/0044523 A1 and US 2002/0077460 A1.

[0106] OX40-L is also referred to as gp34 or ACT-4-L and is expressed onthe surface of select mammalian cells, such as antigen presenting cells.Human OX40-L was initially isolated and described in Miura et al., MolCell Biol 11(3):1313-1325 (1991). U.S. Pat. No. 5,457, which isincorporated by reference in its entirety, describes the murinehomologue of OX40-L.

[0107] OX40-L proteins of the present invention include, but are notlimited to, human OX40-L polypeptides comprising amino acids 1-183 ofSEQ ID NO:26 and polypeptides that comprise N-terminal, C-terminal, orinternal truncations of the foregoing sequences, but retain the desiredbiological activity. DNA sequences encoding such human OX40 polypeptidesinclude, but are not limited to, DNA sequences comprising the nucleotidesequence of SEQ ID NO:25.

[0108] Alternative embodiments provide soluble OX40-L polypeptides.Soluble OX40-L polypeptides comprise all or part of the extracellulardomain of a native OX40 but lack the transmembrane region that wouldcause retention of the polypeptide on a cell membrane. The solubleOX40-L polypeptides retain the ability to bind the OX40. Soluble OX40-Lpolypeptides may also include part of the transmembrane region or partof the cytoplasmic domain or other sequences, provided that the solubleOX40-L protein is capable of being secreted.

[0109] In further embodiments, OX40 antagonists are polypeptidemimetics, such as, but not limited to peptidomimetics, peptibodiesand/or mimotopes developed from the polypeptide sequence of OX40-L (SEQID NO:26) and/or OX40 (SEQ ID NO:28). Polypeptide mimetics may bedeveloped through techniques known in the art, such as combinatorialpeptide libraries. Polypeptide mimetics are peptide-containing moleculeswhich mimic elements of protein secondary structure. A OX40-Lpolypeptide mimetic based on the amino acid sequence of OX40-L will bindto OX40 without activating OX40 and sterically hinder binding ofendogenous OX40-L. A OX40 receptor polypeptide mimetic based on theamino acid sequence of OX40 receptor will bind to OX40-L and stericallyhinder binding of endogenous OX40-L to OX40. OX40-L and OX40 peptidemimetics can be used to antagonize OX40-L binding to OX40 in a subjectand thereby reduce the proinflammatory effects of OX40-L. As such, OX40antagonists in the form of polypeptide mimetics may be used to treatinflammatory and/or immunoregulatory processes associated withcardiovascular disease.

[0110] Other forms of OX40-L and OX40 that are useful in the presentinvention include muteins and variants (also referred to as analogs),such as naturally occurring variants, that are substantially homologousto the native OX40-L (SEQ ID NO:26) or OX40 (SEQ ID NO:28) polypeptidesequences that retain biological activity.

[0111] This invention additionally provides for the use of OX40antagonists in the manufacture of a medicament for the treatment ofcardiovascular disease. This invention further provides for the use ofpolynucleotides encoding OX40 antagonists in the manufacture of amedicament for the treatment of cardiovascular disease.

[0112] F. CD39

[0113] Alternative embodiments of the invention are directed to treatingcardiovascular disease in a subject having cardiovascular diseasecomprising administering soluble CD39 polypeptides in combination withone or more IL-17 antagonists, IL-18 antagonists, 4-1BB antagonists,CD30 antagonists and/or OX40 antagonists.

[0114] The molecular cloning and structural characterization of CD39 ispresented in Maliszewski et al. (J. Immunol. 153:3574, 1994). A cDNAencoding the cell-surface molecule CD39 has been isolated, cloned andsequenced, as described in U.S. patent application Ser. No. 09/835,147,as well as WO 00/23459, which are incorporated by reference in theirentirety. The nucleic acid sequence and predicted amino acid sequenceare shown in SEQ ID NO:29 and SEQ ID NO:30, respectively.

[0115] The present invention provides methods of using soluble forms ofCD39 to treat cardiovascular disease, which were constructed by removingthe amino- and carboxy-terminal transmembrane domains. Soluble CD39retains the capacity of wildtype CD39 to metabolize ATP and ADP atphysiologically relevant concentrations as well as the ability to blockand reverse ADP-induced platelet activation and recruitment, includingplatelet aggregation. The use of soluble forms of CD39 is advantageousbecause purification of the polypeptides from recombinant host cells isfacilitated, and because soluble polypeptides are generally moresuitable than membrane-bound forms for clinical administration. BecauseCD39 inhibits platelet activation and recruitment, and thereforeplatelet aggregation, the present invention provides methods andcompositions for inhibiting formation of a thrombus at a site in amammal at which platelets are inappropriately activated, methods for usein controlling platelet reactivity, thereby regulating the hemostaticand thrombotic processes, and methods of inhibiting and/or reversingplatelet aggregation.

[0116] CD39 contains two putative transmembrane regions, near the aminoand carboxy termini, which may serve to anchor the native protein in thecell membrane. The portion of the molecule between the transmembraneregions is external to the cell. As used herein, the term “CD39polypeptides” includes CD39, homologs of CD39, variants, fragments, andderivatives of CD39, fusion polypeptides comprising CD39, and solubleforms of CD39 polypeptides. The CD39 gene family is reported to containat least four human members: CD39, CD39L2, CD39L3, and CD39L4 (Chadwickand Frischauf, Genomics 50:357, 1998). CD39-L4 is reported to be asecreted apyrase (Mulero et al., J. Biol. Chem. 274(29):20064, 1999).Additional solCD39 variants have been constructed by fusing N-terminalsequences from CD39L2, CD39L3, or CD39L4 to a soluble portion of CD39,as described in detail in U.S. patent application Ser. No. 09/835,147.

[0117] CD39 is an ecto-ADPase (apyrase) located on the surface ofendothelial cells. This enzyme is mainly responsible for the maintenanceof blood fluidity, thus maintaining platelets in the baseline (resting)state. This is accomplished by metabolism of the major platelet agonist,adenosine diphosphate, to adenosine monophosphate, which is not anagonist. Because ADP is the most important agonist of plateletaggregation, and is present in platelet releasate, a substance whichcatabolizes ADP is useful in treating or preventing disease states thatinvolve inappropriate aggregation of platelets.

[0118] Apyrase activity resides in the extracellular domain of CD39.Thus, CD39 polypeptides include soluble forms of CD39 such as thosehaving an amino terminus selected from the group consisting of aminoacids 36-44 of SEQ ID NO:30, and a carboxy terminus selected from thegroup consisting of amino acids 471-478 of SEQ ID NO:30, and whichexhibit CD39 biological activity. Soluble CD39 polypeptides also includethose polypeptides which include part of either or both of thetransmembrane regions, provided that the soluble CD39 polypeptide iscapable of being secreted from a cell, and retains CD39 biologicalactivity. Soluble CD39 polypeptides further include oligomers or fusionpolypeptides comprising the extracellular portion of CD39, and fragmentsof any of these polypeptides that have biological activity.

[0119] The term “biological activity,” with regards to CD39, includesapyrase enzymatic activity as well as the ex vivo and in vivo activitiesof CD39. Apyrases catalyze the hydrolysis of nucleoside tri- and/ordi-phosphates, but a given apyrase may display different relativespecificities for either nucleoside triphosphates or nucleosidediphosphates. Biological activity of soluble forms of CD39 may bedetermined, for example, in an ectonucleotidase or apyrase assay (e.g.ATPase or ADPase assays), or in an assay that measures inhibition ofplatelet aggregation. Exemplary assays are disclosed herein; those ofskill in the art will appreciate that other, similar types of assays canbe used to measure biological activity.

[0120] In further embodiments, CD39 compositions for the treatment ofcardiovascular disease comprise polypeptide mimetics, such as, but notlimited to peptidomimetics, peptibodies and/or mimotopes developed fromthe polypeptide sequence of CD39 (SEQ ID NO:30). Polypeptide mimeticsmay be developed through techniques known in the art, such ascombinatorial peptide libraries. Polypeptide mimetics arepeptide-containing molecules which mimic elements of protein secondarystructure. A CD39 polypeptide mimetic based on the amino acid sequenceof CD39 will catalyze the hydrolysis of nucleoside tri- and/ordi-phosphates. As such, CD39 antagonists in the form of polypeptidemimetics may be used to treat cardiovascular disease.

[0121] Other forms of CD39 that are useful in the present inventioninclude muteins and variants (also referred to as analogs), such asnaturally occurring variants, that are substantially homologous to thenative CD39 (SEQ ID NO:30) polypeptide sequences and as described inU.S. patent application Ser. No. 09/835,147 that retain biologicalactivity.

[0122] This invention additionally provides for the use of CD39 in themanufacture of a medicament for the treatment of cardiovascular disease.This invention further provides for the use of polynucleotides encodingCD39 in the manufacture of a medicament for the treatment ofcardiovascular disease.

[0123] CD39, in all its forms as described herein and in U.S. patentapplication Ser. No. 09/835,147, may be used in combination with one ormore IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonist in the treatmentof cardiovascular disease.

[0124] G. Further Embodiments of IL-17, IL-18, 4-1BB, CD30 and OX40Antagonists and CD39

[0125] Other forms of the IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 that are useful in thepresent invention include muteins and variants (also referred to asanalogs), such as naturally occurring variants, that are substantiallyhomologous to the native sequences provided herein, as well as thesequences provided in the patents incorporated by reference.

[0126] Substantially homologous means a variant amino acid sequence thatis at least 75%, at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98% or at least 99% identical tothe native amino acid sequences, as disclosed above. The percentidentity of two amino acid or two nucleic acid sequences can bedetermined by visual inspection and mathematical calculation, or morepreferably, the comparison is done by comparing sequence informationusing a computer program. An exemplary, preferred computer program isthe Genetics Computer Group (GCG; Madison, Wis.) Wisconsin packageversion 10.0 program, ‘GAP’ (Devereux et al., 1984, Nucl. Acids Res. 12:387). The preferred default parameters for the ‘GAP’ program includes:(1) The GCG implementation of a unary comparison matrix (containing avalue of 1 for identities and 0 for non-identities) for nucleotides, andthe weighted amino acid comparison matrix of Gribskov and Burgess, Nucl.Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds.,Atlas of Polypeptide Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358, 1979; or other comparable comparisonmatrices; (2) a penalty of 30 for each gap and an additional penalty of1 for each symbol in each gap for amino acid sequences, or penalty of 50for each gap and an additional penalty of 3 for each symbol in each gapfor nucleotide sequences; (3) no penalty for end gaps; and (4) nomaximum penalty for long gaps. Other programs used by those skilled inthe art of sequence comparison can also be used, such as, for example,the BLASTN program version 2.0.9, available for use via the NationalLibrary of Medicine website www.ncbi.nlm.nih.gov/gorf/wblast2.cgi, orthe UW-BLAST 2.0 algorithm. Standard default parameter settings forUW-BLAST 2.0 are described at the following Internet site:sapiens.wustl.edu/blast/blast/#Features. In addition, the BLASTalgorithm uses the BLOSUM62 amino acid scoring matix, and optionalparameters that can be used are as follows: (A) inclusion of a filter tomask segments of the query sequence that have low compositionalcomplexity (as determined by the SEG program of Wootton and Federhen(Computers and Chemistry, 1993); also see Wootton and Federhen, 1996,Analysis of compositionally biased regions in sequence databases,Methods Enzymol. 266: 554-71) or segments consisting ofshort-periodicity internal repeats (as determined by the XNU program ofClaverie and States (Computers and Chemistry, 1993)), and (B) astatistical significance threshold for reporting matches againstdatabase sequences, or E-score (the expected probability of matchesbeing found merely by chance, according to the stochastic model ofKarlin and Altschul (1990); if the statistical significance ascribed toa match is greater than this E-score threshold, the match will not bereported.); preferred E-score threshold values are 0.5, or in order ofincreasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, 1e-5,1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100.

[0127] Such variants include polypeptides that are substantiallyhomologous to native IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 sequences, but which havean amino acid sequence different from that of a native IL-17 receptorbecause of one or more deletions, insertions or substitutions.Particular embodiments include, but are not limited to, IL-17, IL-17R,IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L andCD39 polypeptides that comprise at least one conservative amino acidsubstitution. Alternative embodiments comprise IL-17, IL-17R, IL-18,IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39polypeptides comprising from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequences. The IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L,CD30, CD30-L, OX40, OX40-L and CD39 -encoding polynucleotides of thepresent invention include variants that differ from a native IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L and CD39 polynucleotide sequence because of one or moredeletions, insertions or substitutions, but that encode a biologicallyactive polypeptide. Included as variants of IL-17, IL-17R, IL-18,IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39polypeptides are those variants that are naturally occurring, such asallelic forms and alternatively spliced forms, as well as variants thathave been constructed by modifying the amino acid sequence of an IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L and CD39 polypeptide or the nucleotide sequence of a nucleic acidencoding an IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L and CD39 polypeptide.

[0128] As mentioned above, IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 variants may comprise asequence having at least one conservatively substituted amino acid,meaning that a given amino acid residue is replaced by a residue havingsimilar physiochemical characteristics. Alternative embodiments comprise1L-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and CD39 variants that comprise between 1-10, 1-20 or 1-30conservatively substituted sequences. Generally, substitutions for oneor more amino acids present in the native polypeptide should be madeconservatively. Examples of conservative substitutions includesubstitution of amino acids outside of the active domain(s), andsubstitution of amino acids that do not alter the secondary and/ortertiary structure of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39. Examples of conservativesubstitutions include substitution of one aliphatic residue for another,such as Ile, Val, Leu, or Ala for one another, or substitutions of onepolar residue for another, such as between Lys and Arg; Glu and Asp; orGln and Asn. Other such conservative substitutions, for example,substitutions of entire regions having similar hydrophobicitycharacteristics, are well known. Naturally occurring variants are alsoencompassed by the invention. Examples of such variants are proteinsthat result from alternate mRNA splicing events or from proteolyticcleavage of the native protein, wherein the native biological propertyis retained.

[0129] For example, a “conservative amino acid substitution” may involvea substitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis” (see, forexample, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67;Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alaninescanning mutagenesis).

[0130] Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the peptidesequence, or to increase or decrease the affinity of the peptide orvehicle-peptide molecules (see preceding formulae) described herein.Exemplary amino acid substitutions are set forth in Table 1. TABLE 1Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) AsnAsn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg ArgIle (I) Leu, Val, Met, Ala, Leu Phe, Norleucine Leu (L) Norleucine, Ile,Val, Ile Met, Ala, Phe Lys (K) Arg, 1,4 Diamino- Arg butyric Acid, Gln,Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro(P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, PheTyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Leu Ala,Norleucine

[0131] In certain embodiments, conservative amino acid substitutionsalso encompass non-naturally occurring amino acid residues which aretypically incorporated by chemical peptide synthesis rather than bysynthesis in biological systems.

[0132] As noted above, naturally occurring residues may be divided intoclasses based on common sidechain properties that may be useful formodifications of sequence. For example, non-conservative substitutionsmay involve the exchange of a member of one of these classes for amember from another class. Such substituted residues may be introducedinto regions of the peptide that are homologous with non-humanorthologs, or into the non-homologous regions of the molecule. Inaddition, one may also make modifications using P or G for the purposeof influencing chain orientation.

[0133] In making such modifications, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0134] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.(Kyte, et al., J. Mol. Biol., 157: 105-131 (1982)). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±5 are even more particularly preferred.

[0135] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. Thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a biological property of theprotein.

[0136] The following hydrophilicity values have been assigned to aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4). In making changes based upon similar hydrophilicityvalues, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.One may also identify epitopes from primary amino acid sequences on thebasis of hydrophilicity. These regions are also referred to as “epitopiccore regions.”

[0137] A skilled artisan will be able to determine suitable variants ofthe polypeptide as set forth in the foregoing sequences using well knowntechniques. For identifying suitable areas of the molecule that may bechanged without destroying activity, one skilled in the art may targetareas not believed to be important for activity. For example, whensimilar polypeptides with similar activities from the same species orfrom other species are known, one skilled in the art may compare theamino acid sequence of a peptide to similar peptides. With such acomparison, one can identify residues and portions of the molecules thatare conserved among similar polypeptides. It will be appreciated thatchanges in areas of a peptide that are not conserved relative to suchsimilar peptides would be less likely to adversely affect the biologicalactivity and/or structure of the peptide. One skilled in the art wouldalso know that, even in relatively conserved regions, one may substitutechemically similar amino acids for the naturally occurring residueswhile retaining activity (conservative amino acid residuesubstitutions). Therefore, even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the peptide structure.

[0138] Additionally, one skilled in the art can reviewstructure-function studies identifying residues in similar peptides thatare important for activity or structure. In view of such a comparison,one can predict the importance of amino acid residues in a peptide thatcorrespond to amino acid residues that are important for activity orstructure in similar peptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of the peptides.

[0139] One skilled in the art can also analyze the three-dimensionalstructure and amino acid sequence in relation to that structure insimilar polypeptides. In view of that information, one skilled in theart may predict the alignment of amino acid residues of a peptide withrespect to its three dimensional structure. One skilled in the art maychoose not to make radical changes to amino acid residues predicted tobe on the surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. The variants can thenbe screened using activity assays know to those skilled in the art. Suchdata could be used to gather information about suitable variants. Forexample, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

[0140] A number of scientific publications have been devoted to theprediction of secondary structure. See, Moult J., Curr. Op. in Biotech.,7(4): 422-427 (1996), Chou et al., Biochemistry, 13(2): 222-245 (1974);Chou et al., Biochemistry, 113(2): 211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47: 45-148 (1978); Chou et al., Ann.Rev. Biochem., 47: 251-276 and Chou et al., Biophys. J., 26: 367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holm,et al., Nuci. Acid. Res., 27(1): 244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will gain dramatically in accuracy.

[0141] Additional methods of predicting secondary structure include“threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87 (1997);Sippl, et al., Structure, 4(1): 15-9 (1996)), “profile analysis” (Bowie,et al., Science, 253: 164-170 (1991); Gribskov, et al., Meth. Enzym.,183: 146-159 (1990); Gribskov, et al., Proc. Nat. Acad. Sci., 84(13):4355-8 (1987)), and “evolutionary linkage” (See Holm, supra, andBrenner, supra).

[0142] Embodiments of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 variants include IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L and CD39 variants include polypeptide sequences that are at leastabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical in aminoacid sequence to the respective amino acid sequence for IL-17, IL-17R,IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L andCD39, as described above.

[0143] Further modifications in the IL-17, IL-17R, IL-18, IL-18R,IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 polypeptideor IL-17, IL-17R, 1L-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and CD39 polynucleotide sequences can be made by thoseskilled in the art using known techniques. Modifications of interest inthe polypeptide sequences can include the alteration, substitution,replacement, insertion or deletion of a selected amino acid. Forexample, one or more of the cysteine residues can be deleted or replacedwith another amino acid to alter the conformation of the molecule, analteration which may involve preventing formation of incorrectintramolecular disulfide bridges upon folding or renaturation.Techniques for such alteration, substitution, replacement, insertion ordeletion are well known to those skilled in the art (see, e.g., U.S.Pat. No. 4,518,584). As another example, N-glycosylation sites in theIL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and CD39 extracellular domain can be modified to precludeglycosylation, allowing expression of a reduced carbohydrate analog inmammalian and yeast expression systems. N-glycosylation sites ineukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr.Appropriate substitutions, additions, or deletions to the nucleotidesequence encoding these triplets will result in prevention of attachmentof carbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid,for example, is sufficient to inactivate an N-glycosylation site.Alternatively, the Ser or Thr can by replaced with another amino acid,such as Ala. Procedures for inactivating N-glycosylation sites inpolypeptides are known in the art and include, for example, thosedescribed in U.S. Pat. No. 5,071,972. Additional variants within thescope of the invention include IL-17, IL-17R, IL-18, IL-18R, IL-18BP,4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 polypeptides thatcan be modified to create derivatives thereof by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like. Covalentderivatives can be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Preferably, such alteration, substitution,replacement, insertion or deletion does not diminish the biologicalactivity of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L or CD39. One example is a variant that binds withessentially the same binding affinity as does the native form. Bindingaffinity can be measured by conventional procedures, e.g., as describedin U.S. Pat. No. 5,512,457 and as set forth herein. Furthermore, IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L and CD39 molecules may be modified by the addition of one or morewater-soluble polymers, such as, but not limited to, polyethylene glycolto increase bio-availability and/or pharmacokinetic half-life.

[0144] Various means for attaching chemical moieties useful for increasebio-availability and/or pharmacokinetic half-life are currentlyavailable, see, e.g., Patent Cooperation Treaty (“PCT”) InternationalPublication No. WO 96/11953, entitled “N-Terminally Chemically ModifiedProtein Compositions and Methods,” herein incorporated by reference inits entirety. This PCT publication discloses, among other things, theselective attachment of water soluble polymers to the N-terminus ofproteins.

[0145] A preferred polymer vehicle is polyethylene glycol (PEG). The PEGgroup may be of any convenient molecular weight and may be linear orbranched. The average molecular weight of the PEG will preferably rangefrom about 2 kiloDalton (“kD”) to about 100 kD, more preferably fromabout 5 kD to about 50 kD, most preferably from about 5 kD to about 10kD. The PEG groups will generally be attached to the compounds of theinvention via acylation or reductive alkylation through a reactive groupon the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to areactive group on the inventive compound (e.g., an aldehyde, amino, orester group).

[0146] A useful strategy for the PEGylation of synthetic peptidesconsists of combining, through forming a conjugate linkage in solution,a peptide and a PEG moiety, each bearing a special functionality that ismutually reactive toward the other. The peptides can be easily preparedwith conventional solid phase synthesis. The peptides are “preactivated”with an appropriate functional group at a specific site. The precursorsare purified and fully characterized prior to reacting with the PEGmoiety. Ligation of the peptide with PEG usually takes place in aqueousphase and can be easily monitored by reverse phase analytical HPLC. ThePEGylated peptides can be easily purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

[0147] Polysaccharide polymers are another type of water-soluble polymerwhich may be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by α1-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kD to about 70 kD. Dextran is a suitable water solublepolymer for use in the present invention as a vehicle by itself or incombination with another vehicle (e.g., Fc). See, for example, WO96/11953 and WO 96/05309. The use of dextran conjugated to therapeuticor diagnostic immunoglobulins has been reported; see, for example,European Patent Publication No. 0 315 456, which is hereby incorporatedby reference in its entirety. Dextran of about 1 kD to about 20 kD ispreferred when dextran is used as a vehicle in accordance with thepresent invention.

[0148] Additional IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L,CD30, CD30-L, OX40, OX40-L and CD39 derivatives include covalent oraggregative conjugates of the polypeptides with other polypeptides orpolypeptides, such as by synthesis in recombinant culture as N-terminalor C-terminal fusions. Examples of fusion polypeptides are discussedbelow in connection with oligomers. Further, fusion polypeptides cancomprise peptides added to facilitate purification and identification.Such peptides include, for example, poly-His or the antigenicidentification peptides described in U.S. Pat. No. No. 5,011,912 and inHopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG®octapeptide (SEQ ID NO:31), which is highly antigenic and provides anepitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinantpolypeptide. A murine hybridoma designated 4E11 produces a monoclonalantibody that binds the FLAG® peptide in the presence of certaindivalent metal cations, as described in U.S. Pat. No. 5,011,912. The4E11 hybridoma cell line has been deposited with the American TypeCulture Collection under accession no. HB 9259. Monoclonal antibodiesthat bind the FLAG® peptide are available from Eastman Kodak Co.,Scientific Imaging Systems Division, New Haven, Conn.

[0149] Additional embodiments of IL-17, IL-17R, IL-18, IL-18R, IL-18BP,4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 that may be used inthe methods described herein include oligomers or fusion polypeptidesthat contain IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L,CD30, CD30-L, OX40, OX40-L and/or CD39 polypeptide, one or morefragments of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L,CD30, CD30-L, OX40, OX40-L and/or CD39, or any of the derivative orvariant forms of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L,CD30, CD30-L, OX40, OX40-L and/or CD39 as disclosed herein, as well asin the U.S. patents listed above. In particular embodiments, theoligomers comprise soluble IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and/or CD39 polypeptides. Oligomerscan be in the form of covalently linked or non-covalently-linkedmultimers, including dimers, trimers, or higher oligomers. Inalternative embodiments, IL-17, 1L-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 oligomers comprise multipleIL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and CD39 polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the polypeptides, suchpeptides having the property of promoting oligomerization. Leucinezippers and certain polypeptides derived from antibodies are among thepeptides that can promote oligomerization of the polypeptides attachedthereto, as described in more detail below.

[0150] Immunoglobulin-based Oligomers. Suitable forms of IL-17, IL-17R,IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L andCD39 antagonists include chimeric proteins which include a secondpolypeptide that may promote the spontaneous formation by the chimericprotein of a dimer, trimer or higher order multimer that is capable ofbinding their respective cognates and thereby inhibiting or reducing theeffects of inflammation and symptoms of cardiovascular disease. Chimericproteins used as antagonists may be proteins that contain portions of anantibody molecule and a soluble polypeptide from IL-17, IL-17R, IL-18,IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX 40, OX40-L and/orCD39. Suitable fusion proteins include an IL-17, IL-17R, IL-18, IL-18R,IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39polypeptide, e.g. the extracellular domain, or a fragment of theextracellular domain, linked to an immunoglobulin Fc region. Fragmentsof a Fc region may also be used, as well as Fc muteins that exhibitdecreased affinity for Fc receptors. Soluble IL-17, IL-17R, IL-18,IL-18R, IL-18BP, 4-1BB, 4-IBB-L, CD30, CD30-L, OX40, OX40-L and CD39, aswell as fragments thereof, can be fused directly or through linkersequences to the Fc portion of an immunoglobulin.

[0151] One embodiment of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 antagonist is directed to adimer comprising two fusion polypeptides created by fusing an IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L and/or CD39 polypeptide to a Fc polypeptide derived from anantibody. A gene fusion encoding such a fusion polypeptide is insertedinto an appropriate expression vector. IL-17, IL-17R, IL-18, IL-18R,IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39-Fc-fusionpolypeptides are expressed in host cells transformed with therecombinant expression vector, and allowed to assemble much likeantibody molecules, whereupon interchain disulfide bonds form betweenthe Fc moieties to yield divalent molecules. One suitable Fcpolypeptide, described in PCT application WO 93/10151, is a single chainpolypeptide extending from the N-terminal hinge region to the nativeC-terminus of the Fc region of a human IgG1 antibody. For a bivalentform of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L or CD39, such a fusion could be to the Fc portionof an IgG molecule. Other immunoglobulin isotypes can also be used togenerate such fusions. For example, a polypeptide-IgM fusion wouldgenerate a decavalent form of the polypeptide of the invention.

[0152] The term “Fc polypeptide” as used herein includes native andmutein forms of polypeptides made up of the Fc region of an antibodycomprising any or all of the CH domains of the Fc region. Truncatedforms of such polypeptides containing the hinge region that promotesdimerization are also included. Preferred Fc polypeptides comprise an Fcpolypeptide derived from a human IgG1 antibody. As one alternative, anoligomer is prepared using polypeptides derived from immunoglobulins.Preparation of fusion polypeptides comprising certain heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) are known in the art and have been described,e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature344:677, 1990); and Hollenbaugh and Aruffo (“Construction ofImmunoglobulin Fusion Polypeptides”,in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11, 1992). Another useful Fc polypeptideis the Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum etal., (EMBO J. 13:3992-4001, 1994). The amino acid sequence of thismutein is identical to that of the native Fc sequence presented in WO93/10151, except that amino acid 19 has been changed from Leu to Ala,amino acid 20 has been changed from Leu to Glu, and amino acid 22 hasbeen changed from Gly to Ala. The mutein exhibits reduced affinity forFc receptors. The above-described fusion polypeptides comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Polypeptide A orPolypeptide G columns. In other embodiments, the polypeptides of theinvention can be substituted for the variable portion of an antibodyheavy or light chain. If fusion polypeptides are made with both heavyand light chains of an antibody, it is possible to form an oligomer withas many as four IL-17R extracellular regions.

[0153] Peptide-linker Based Oligomers. Alternatively, the oligomer is afusion polypeptide comprising multiple IL-17, IL-17R, IL-18, IL-18R,IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and/or CD39polypeptides, with or without peptide linkers (spacer peptides). Amongthe suitable peptide linkers are those described in U.S. Pat. Nos.4,751,180 and 4,935,233. A DNA sequence encoding a desired peptidelinker can be inserted between, and in the same reading frame as, theDNA sequences of the invention, using any suitable conventionaltechnique. For example, a chemically synthesized oligonucleotideencoding the linker can be ligated between the sequences. In particularembodiments, a fusion polypeptide comprises from two to four solubleIL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and/or CD39 polypeptides, separated by peptide linkers.Suitable peptide linkers, their combination with other polypeptides, andtheir use are well known by those skilled in the art.

[0154] Oligomeric forms of IL-17, IL-18, 4-1BB, CD30, OX40 and CD39antagonists suitable for use in treating cardiovascular disease alsoinclude an IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L and/or CD39 polyeptide, the extracellular domain ofan IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and/or CD39 polypeptide, or an IL-17, IL-17R, IL-18,IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39antagonistic fragment of the extracellular domain associated with azipper domain, such as zipper proteins described in U.S. Pat. No.5,716,805, the disclosure of which is incorporated by reference herein.Other Examples of zipper domains are those found in the yeasttranscription factor GCN4 and a heat-stable DNA-binding protein found inrat liver (C/EBP; Landschulz et al., Science 243:1681, 1989), thenuclear transforming proteins, fos and jun, which preferentially form aheterodimer (O'Shea et al., Science 245:646, 1989; Turner and Tjian,Science 243:1689, 1989), and the gene product of the murineproto-oncogene, c-myc (Landschulz et al., Science 240:1759, 1988). Thefusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess leucine zipper domains (Buckland and Wild, Nature 338:547, 1989;Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research andHuman Retroviruses 6:703, 1990). Leucine zipper domains are peptidesthat promote oligomerization of the polypeptides in which they arefound. Leucine zippers were originally identified in several DNA-bindingpolypeptides and have since been found in a variety of differentpolypeptides. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. The zipperdomain (also referred to herein as an oligomerizing, oroligomer-forming, domain) comprises a repetitive heptad repeat, oftenwith four or five leucine residues interspersed with other amino acids.Use of leucine zippers and preparation of oligomers using leucinezippers are well known in the art.

[0155] The present invention comprises fusion polypeptides with orwithout spacer amino acid linking groups. For example, two solubleIL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and/or CD39 domains can be linked with a linker sequence,such as (Gly)₄Ser(Gly)₅Ser (SEQ ID NO:32), which is described in U.S.Pat. No. 5,073,627. Other linker sequences include, for example,GlyAlaGlyGlyAlaGlySer(Gly)₅Ser (SEQ ID NO:33), (Gly₄Ser)₂ (SEQ IDNO:34), (GlyThrPro)₃ (SEQ ID NO:35), and (Gly₄Ser)₃Gly₄SerGly₅Ser (SEQID NO:36).

[0156] Nucleic acid sequences encoding soluble IL-17, IL-17R, IL-18,IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39polypeptides having altered glycosylation sites, deleted or substitutedCys residues, or modified proteolytic cleavage sites, nucleic acidsequences encoding sub-units of IL-17, IL-17R, IL-18, IL-18R, IL-18BP,4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 polypeptides orfusion polypeptides of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 with other peptides,allelic variants of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39, mammalian homologs ofIL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and CD39, and nucleic acid sequences encoding IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L and CD39 polypeptides derived from alternative mRNA constructs,or those that encode peptide having substituted or additional aminoacids, are examples of nucleic acid sequences according to theinvention.

[0157] Due to degeneracy of the genetic code, there can be considerablevariation in nucleotide sequences encoding the same amino acid sequence.Embodiments include sequences capable of hybridizing under moderatelystringent conditions. The basic parameters affecting the choice ofhybridization conditions and guidance for devising suitable conditionsare set forth by Sambrook, Fritsch, and Maniatis (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., chapters 9 and 11; and Current Protocols inMolecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc.,sections 2.10 and 6.3-6.4), and can be readily determined by thosehaving ordinary skill in the art based on, for example, the lengthand/or base composition of the DNA. One way of achieving moderatelystringent conditions involves the use of a prewashing solutioncontaining 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization bufferof about 50% formamide, 6×SSC, and a hybridization temperature of about55 degrees C. (or other similar hybridization solutions, such as onecontaining about 50% formamide, with a hybridization temperature ofabout 42 degrees C.), and washing conditions of about 60 degrees C., in0.5×SSC, 0.1% SDS. Generally, highly stringent conditions are defined ashybridization conditions as above, but with washing at approximately 68degrees C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mMNaH.sub.2 PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC(1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization andwash buffers; washes are performed for 15 minutes after hybridization iscomplete. It should be understood that the wash temperature and washsalt concentration can be adjusted as necessary to achieve a desireddegree of stringency by applying the basic principles that governhybridization reactions and duplex stability, as known to those skilledin the art and described further below (see, e.g., Sambrook et al.,1989). When hybridizing a nucleic acid to a target nucleic acid ofunknown sequence, the hybrid length is assumed to be that of thehybridizing nucleic acid. When nucleic acids of known sequence arehybridized, the hybrid length can be determined by aligning thesequences of the nucleic acids and identifying the region or regions ofoptimal sequence complementarity. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be 5to 10.degrees C. less than the melting temperature (Tm) of the hybrid,where Tm is determined according to the following equations. For hybridsless than 18 base pairs in length, Tm (degrees C.)=2(# of A+T bases)+4(#of #G+C bases). For hybrids above 18 base pairs in length, Tm (degreesC.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165M). Preferably, each suchhybridizing nucleic acid has a length that is at least 15 nucleotides(or more preferably at least 18 nucleotides, or at least 20 nucleotides,or at least 25 nucleotides, or at least 30 nucleotides, or at least 40nucleotides, or most preferably at least 50 nucleotides), or at least25% (more preferably at least 50%, or at least 60%, or at least 70%, andmost preferably at least 80%) of the length of the nucleic acid of thepresent invention to which it hybridizes, and has at least 60% sequenceidentity (more preferably at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%,and most preferably at least 99.5%) with the nucleic acid of the presentinvention to which it hybridizes, where sequence identity is determinedby comparing the sequences of the hybridizing nucleic acids when alignedso as to maximize overlap and identity while minimizing sequence gaps asdescribed in more detail above.

[0158] In alternative embodiments, IL-17, IL-17R, IL-18, IL-18R,IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and/or CD39polynucleotides include those that encode polypeptides that are at least75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%,or at least 96%, or at least 97%, or at least 98%, or at least 99%identical in amino acid sequence to the amino acid sequence of native orat least 80% polypeptide sequences as set forth above and in thesequence listing. For polynucleotides that encode a fragment of IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L and/or CD39, percent identity of the fragment is based on percentidentity to the corresponding portion of full-length IL-17, IL-17R,IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-Land/or CD39 polypeptide, respectively.

[0159] Mutations can be introduced into nucleic acids by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes a variant havingthe desired amino acid insertion, substitution, or deletion.

[0160] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, Jan. 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, andare incorporated by reference herein.

[0161] Polynucleotide sequences that encode IL-17 receptor polypeptidescomprising various additions or substitutions of amino acid residues orsequences, or deletions of terminal or internal residues or sequencesnot needed for biological activity can be prepared. For example,N-glycosylation sites can be modified to preclude glycosylation whileallowing expression of a homogeneous, reduced carbohydrate variant usingyeast expression systems. N-glycosylation sites in eukaryoticpolypeptides are characterized by an amino acid triplet Asn-X-Y, whereinX is any amino acid except Pro and Y is Ser or Thr. Appropriatemodifications to the nucleotide sequence encoding this triplet willresult in substitutions, additions or deletions that prevent attachmentof carbohydrate residues at the Asn side chain.

[0162] In another example, sequences encoding Cys residues can bealtered to cause the Cys residues to be deleted or replaced with otheramino acids, preventing formation of incorrect intramolecular disulfidebridges upon renaturation. Thus, Cys residues may be replaced withanother amino acid or deleted without affecting polypeptide tertiarystructure or disulfide bond formation.

[0163] Other approaches to mutagenesis involve modification of sequencesencoding dibasic amino acid residues to enhance expression in yeastsystems in which KEX2 protease activity is present. Other variants areprepared by modification of adjacent dibasic amino acid residues, toenhance expression in yeast systems in which KEX2 protease activity ispresent. EP 212,914 discloses the use of site-specific mutagenesis toinactivate KEX2 protease processing sites in a polypeptide. KEX2protease processing sites are inactivated by deleting, adding orsubstituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs toeliminate the occurrence of these adjacent basic residues. Similarmodification may be made to sequences encoding sites recognized andcleaved by other proteolytic enzymes. Sub-units of a IL-17, IL-17R,IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-Land/or CD39 polypeptide may be constructed by deleting sequencesencoding terminal or internal residues or sequences not necessary forbiological activity. Sequences encoding fusion polypeptides as describedbelow may be constructed by ligating sequences encoding additional aminoacid residues to the inventive sequences without affecting biologicalactivity.

[0164] Mutations in nucleotide sequences constructed for expression of asoluble IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L and/or CD39 must, of course, preserve the readingframe phase of the coding sequences and preferably will not createcomplementary regions that could hybridize to produce secondary mRNAstructures such as loops or hairpins which would adversely affecttranslation of the receptor mRNA. Although a mutation site may bepredetermined, it is not necessary that the nature of the mutation perse be predetermined. For example, in order to select for optimumcharacteristics of mutants at a given site, random mutagenesis may beconducted at the target codon and the expressed mutated polypeptidesscreened for the desired activity.

[0165] Not all mutations in the nucleotide sequence which encodes aIL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and/or CD39 polypeptide will be expressed in the finalproduct, for example, nucleotide substitutions may be made to enhanceexpression, primarily to avoid secondary structure loops in thetranscribed mRNA or to provide codons that are more readily translatedby the selected host, e.g., the well-known E. coli preference codons forE. coli expression.

[0166] In the genome, IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and/or CD39 polypeptides are encodedby multi-exon genes. The present invention further includes alternativemRNA constructs that can be attributed to different mRNA splicing eventsfollowing transcription and which hybridize with the cDNAs disclosedherein under conditions of moderate stringency, as defined above.

[0167] H. Antibodies as IL-17, IL-18, 4-1BB, CD30 and OX40 Antagonists

[0168] IL-17, IL-18, 4-1BB, CD30 and OX40 antagonists include antibodiesthat specifically bind IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40 or OX40-L. More specifically, IL-17antagonists include antibodies directed against IL-17 that specificallybind IL-17 and partially or completely inhibit binding of IL-17 to IL-17receptor, and antibodies directed against IL-17 receptor thatspecifically bind IL-17 receptor and inhibit binding of IL-17 withoutthemselves activating the IL-17 receptor; IL-18 antagonists includeantibodies directed against IL-18 that specifically bind IL-18 andpartially or completely inhibit binding of IL-18 to IL-18R; antibodiesdirected against IL-18R that specifically bind IL-18R and inhibitreceptor binding of IL-18 without themselves transducing a signal viaIL-18R; 4-1BB antagonists include antibodies directed against 4-1BB thatspecifically bind 4-1BB and partially or completely inhibit binding of4-1BB to 4-1BB-L; antibodies directed against 4-1BB that specificallybind 4-1BB and inhibit binding of 4-1BB-L without themselves transducinga signal via 4-1BB; CD30 antagonists include antibodies directed againstCD30-L that specifically bind CD30-L and partially or completely inhibitbinding of CD30-L to CD30; antibodies directed against CD30 thatspecifically bind CD30 and inhibit binding of CD30-L without themselvesactivating the CD30; and, OX40 antagonists include antibodies directedagainst OX40-L that specifically bind OX40-L and partially or completelyinhibit binding of OX40-L to OX40; antibodies directed against OX40 thatspecifically bind OX40 and inhibit binding of OX40-L without themselvesactivating the OX40.

[0169] IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40 or OX40-L, as well as fragments, variants, muteins,derivatives and fusion proteins thereof, as set forth above, can beemployed as “immunogens” in producing antibodies that may be used in thediagnosis and treatment of cardiovascular disease. In making IL-17,IL-18, 4-1BB, CD30 and OX40 antagonists in the form of antibodies, whenreference is made to IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40 or OX40-L it is understood to also encompassfragments, variants, muteins, derivatives and fusion proteins thereof. Anumber of antibodies have been made to IL-17, IL-17R, IL-18, IL-18R,4-1BB, 4-1BB-L, OX40, CD30, CD30-L and CD39, as shown in Table 3 ofExample 6.

[0170] IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L and CD39 contain antigenic determinants or epitopesthat elicit the formation of antibodies. These antigenic determinants orepitopes can be either linear or conformational (discontinuous). Linearepitopes are composed of a single section of amino acids of thepolypeptide, while conformational or discontinuous epitopes are composedof amino acids sections from different regions of the polypeptide chainthat are brought into close proximity upon polypeptide folding. Epitopescan be identified by any of the methods known in the art. Additionally,epitopes from the polypeptides of the invention can be used as researchreagents, in assays, and to purify specific binding antibodies fromsubstances such as polyclonal sera or supernatants from culturedhybridomas. Such epitopes or variants thereof can be produced usingtechniques known in the art such as solid-phase synthesis, chemical orenzymatic cleavage of a polypeptide, or using recombinant DNAtechnology.

[0171] Antibodies to IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39 can conveniently begenerated against a recombinantly produced form of the proteinsdescribed above and provided in the respective sequence identifiernumbers. IL-17, IL-18, 4-1BB, CD30 and OX40 antagonists that areantibodies include but are not limited to polyclonal antibodies,monoclonal antibodies (mAbs), humanized or chimeric antibodies, singlechain antibodies, Fab fragments, F(ab′)₂ fragments, fragments producedby a Fab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. Such antibodies can beutilized in methods of treating cardiovascular disease.

[0172] Both polyclonal and monoclonal antibodies to IL-17, IL-17R,IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L andCD39 can be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler andMilstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridomatechnique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al.,Proc. Natl. Acad. Sci. USA 80:2026, 1983); and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96).

[0173] Methods of making humanized monoclonal antibodies are well known,and include for example those described in U.S. Pat. No. 5,585,089(Protein Design: C L Queen et al.; “Humanized Immunoglobulins”), U.S.Pat. No. 5,565,332 (“Production of Chimeric Antibodies-A CombinatorialApproach”), U.S. Pat. No. 5,225,539 (Med Res Council: G P Winter;“Recombinant Altered Antibodies And Methods Of Making AlteredAntibodies”), U.S. Pat. No. 5,693,761-762 (Protein Design: C L Queen etal.; “Polynucleotides Encoding Improved Humanized Immunoglobulins”,and“Humanized Immunoglobulins”), and U.S. Pat. No. 5,530,101 (ProteinDesign: C L Queen et al.; “Humanized Immunoglobulins”), and referencescited therein.

[0174] Hybridoma cell lines that produce monoclonal antibodies specificfor IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L and CD39 are contemplated herein. Such hybridomas can beproduced and identified by conventional techniques. For the productionof antibodies, various host animals may be immunized by injection with1L-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD 30, CD30-L,OX40, OX40-L or CD39 polypeptide that is immunogenic. Such host animalsmay include, but are not limited to, horse, goat, sheep, cow, rabbits,mice, and rats, to name a few. Various adjuvants may be used to increasethe immunological response. Depending on the host species, suchadjuvants include, but are not limited to, Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. The monoclonal antibodies can be recovered byconventional techniques. Such monoclonal antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclassthereof. The hybridoma producing the mAb may be cultivated in vitro orin vivo. Or, the antibody genes can be cloned and optionally otherwisealtered, and expressed in another cell line approved for recombinantproduction of protein pharmaceuticals such as, for example, CHO cells.

[0175] Alternatively, libraries of antibody fragments can be screenedand used to develop human antibodies through recombinant techniques.Such libraries are commercially available from, for example, CambridgeAntibody Technology (Melbourne, UK), and Morphosys (Munich, Del.).

[0176] In addition, techniques developed for the production of “chimericantibodies” (Takeda et al., Nature, 314:452, 1985) by splicing the genesfrom a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a porcine mAb and a humanimmunoglobulin constant region. The monoclonal antibodies of theinvention also include humanized versions of murine monoclonalantibodies. Such humanized antibodies can be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. For example, transgenic mice intowhich genetic material encoding one or more human immunoglobulin chainshas been introduced may be employed. Such mice may be geneticallyaltered in a variety of ways. The genetic manipulation may result inhuman immunoglobulin polypeptide chains replacing endogenousimmunoglobulin chains in at least some (preferably virtually all)antibodies produced by the animal upon immunization. Procedures for theproduction of chimeric and further engineered monoclonal antibodiesinclude those described in Riechmann et al. (Nature 332:323, 1988), Liuet al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934,1989), and Winter and Harris (TIPS 14:139, Can, 1993). Procedures togenerate antibodies transgenically can be found in GB 2,272,440, U.S.Pat. Nos. 5,569,825 and 5,545,806 and related patents claiming prioritytherefrom, all of which are incorporated by reference herein. For use inhumans, the antibodies are typically human or humanized; techniques forcreating such human antibodies are also known. Transgenic animals formaking human antibodies are available from, for example, Medarex Inc.(Princeton, N.J.) Protein Design Labs, Inc. (Fremont, Calif.) andAbgenix Inc. (Fremont, Calif.).

[0177] Expression of a humanized immunoglobulin sequences in bacterialhosts may be used to select higher affinity humanized immunoglobulinsequences by mutagenizing the CDR regions and producing bacteriophagedisplay libraries which may be screened for humanized immunoglobulin CDRvariants which possess high affinity and/or high specificity binding toIL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L or CD39. One potential advantage of such affinitysharpening is the generation of humanized immunoglobulin CDR variantsthat have improved binding affinity and/or reduced cross-reactivity withmolecules other than the molecule to which they were raised. Methods forproducing phage display libraries having immunoglobulin variable regionsequences are provided in the art (see, e.g., Cesareni, FEBS Lett307:66, 1992; Swimmer et al., Proc. Natl. Acad. Sci. USA 89:3756, 1992;Gram et al., Proc. Natl. Acad. Sci. USA 89:3576, 1992; Clackson et al.,Nature 352:624, 1991; Scott & Smith, Science 249:386, 1990; Garrard etal., Bio/Techniques 9:1373, 1991; which are incorporated herein byreference in their entirety for all purposes. The resultant affinitysharpened CDR variant humanized immunoglobulin sequences aresubsequently expressed in a suitable host.

[0178] Antibody fragments, which recognize specific epitopes, may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the (ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., Science, 246:1275, 1989) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778; Bird, Science 242:423, 1988; Huston et al., Proc.Natl. Acad. Sci. USA 85:5879, 1988; and Ward et al., Nature 334:544,1989) can also be adapted to produce single chain antibodies againstpolypeptides containing IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 amino acid sequences. Inaddition, antibodies to the IL-17, IL-17R, IL-18, IL-18R, IL-18BP,4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 polypeptides can, inturn, be utilized to generate anti-idiotype antibodies using techniquesknown to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J7(5):437, 1993; and Nissinoff, J. Immunol. 147(8):2429, 1991).

[0179] I. Nucleic Acid-Based IL-17, IL-18, 4-1BB, CD30 and OX40Antagonists

[0180] In alternative embodiments, nucleic acid-based immuno therapy canbe designed to reduce the level of endogenous IL-17, IL-17R, IL-18,IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 geneexpression, e.g., using antisense or ribozyme approaches to inhibit orprevent translation of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 mRNA transcripts; triplehelix approaches to inhibit transcription of the IL-17, IL-17R, IL-18,IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39gene; or targeted homologous recombination to inactivate or “knock out”the IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L,OX40, OX40-L or CD39 gene or its endogenous promoter.

[0181] Antisense RNA and DNA molecules act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingpolypeptide translation. Antisense approaches involve the design ofoligonucleotides (either DNA or RNA) that are complementary to a mRNAhaving an IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L or CD39 polynucleotide sequence. Absolutecomplementarity, although preferred, is not required. A sequence“complementary” to a portion of RNA, as referred to herein, means asequence having sufficient complementarity to be able to hybridize withthe RNA, thereby forming a stable duplex. Oligonucleotides that arecomplementary to the 5′ end of the message, e.g., the 5′ untranslatedsequence up to and including the AUG initiation codon, should work mostefficiently at inhibiting translation. However, oligonucleotidescomplementary to either the 5′- or 3′- non-translated, non-codingregions of the IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L,CD30, CD30-L, OX40, OX40-L or CD39 gene transcript could be used in anantisense approach to inhibit translation of endogenous IL-17, IL-17R,IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L orCD39. Oligonucleotides complementary to the 5′ untranslated region ofthe mRNA should include the complement of the AUG start codon. Antisensenucleic acids should be at least six nucleotides in length, and arepreferably oligonucleotides ranging from 6 to about 50 nucleotides inlength. The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, and the like. The oligonucleotide mayinclude other appended groups such as peptides (e.g., for targeting hostcell receptors in vivo), or agents facilitating transport across thecell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988), orhybridization-triggered cleavage agents or intercalating agents (see,e.g., Zon, 1988, Pharm. Res. 5:539-549).

[0182] The antisense molecules are delivered to cells, which express atranscript having an IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 polynucleotide sequence invivo by, for example, injecting directly into the tissue or cellderivation site, or by use of modified antisense molecules, designed totarget the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systemically. Another approachutilizes a recombinant DNA construct in which the antisenseoligonucleotide is placed under the control of a strong pol III or polII promoter. The use of such a construct to transfect target cells inthe subject will result in the transcription of sufficient amounts ofsingle stranded RNAs that will form complementary base pairs with theendogenous IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L or CD39 transcripts and thereby prevent translationof the IL-17 mRNA. For example, a vector can be introduced in vivo suchthat it is taken up by a cell and directs the transcription of anantisense RNA. Such a vector can remain episomal or become chromosomallyintegrated, so long as it can be transcribed to produce the desiredantisense RNA. Vectors can be plasmid, viral, or others known in theart, used for replication and expression in mammalian cells.

[0183] Ribozyme molecules designed to catalytically cleave mRNAtranscripts having an IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 polynucleotide sequenceprevent translation of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 mRNA (see, e.g., PCTInternational Publication WO90/11364, published Oct. 4, 1990; U.S. Pat.No. 5,824,519). Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. A major advantage of this approach isthat, because they are sequence-specific, only mRNAs with particularsequences are inactivated. There are two basic types of ribozymesnamely, tetrahymena-type (Hasselhoff, Nature, 334:585-591, 1988) and“hammerhead”-type. Tetrahymena-type ribozymes recognize sequences, whichare four bases in length, while “hammerhead”-type ribozymes recognizebase sequences 11-18 bases in length. The longer the recognitionsequence, the greater the likelihood that the sequence will occurexclusively in the target mRNA species. Consequently, hammerhead-typeribozymes are preferable to tetrahymena-type ribozymes.

[0184] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g. for improved stability, targeting, andthe like). A typical method of delivery involves using a DNA construct“encoding” the ribozyme under the control of a strong constitutive polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L or CD39 message and inhibit translation. Because ribozymes,unlike antisense molecules, are catalytic, a lower intracellularconcentration is required for efficiency.

[0185] Alternatively, endogenous IL-17, IL-17R, IL-18, IL-18R, IL-18BP,4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 expression can bereduced by targeting deoxyribonucleotide sequences complementary to theregulatory region of the target gene (i.e., the target gene promoterand/or enhancers) to form triple helical structures that preventtranscription of the target IL-17, IL-17R, IL-18, IL-18R, IL-18BP,4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 gene (see generally,Helene, 1991, Anticancer Drug Des., 6(6), 569-584; Helene, et al., 1992,Ann. N.Y. Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12),807-815).

[0186] Antisense RNA and DNA, ribozyme, and triple helix molecules ofthe invention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules and include techniques for chemicallysynthesizing oligodeoxyribonucleotides and oligoribonucleotides such as,for example, solid phase phosphoramidite chemical synthesis, e.g. by useof an automated DNA synthesizer (such as are commercially available fromBiosearch, Applied Biosystems, and the like). As examples,phosphorothioate oligonucleotides may be synthesized by the method ofStein et al., 1988, Nucl. Acids Res. 16:3209. Methylphosphonateoligonucleotides can be prepared by use of controlled pore glass polymersupports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:7448-7451). Alternatively, RNA molecules may be generated by in vitroand in vivo transcription of DNA sequences encoding the antisense RNAmolecule. Such DNA sequences may be incorporated into a wide variety ofvectors that incorporate suitable RNA polymerase promoters such as theT7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructsthat synthesize antisense RNA constitutively or inducibly, depending onthe promoter used, can be introduced stably into cell lines.

[0187] In alternative embodiments IL-17, IL-17R, IL-18, IL-18R, IL-18BP,4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39 expression may beblocked by post-translational gene silencing, such as by double-strandedRNA-induced gene silencing, also known as RNA interference (RNAi). RNAsequences of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L,CD30, CD30-L, OX40, OX40-L or CD39 may be modified to providedouble-stranded sequences or short hairpin RNAs for therapeutic use.

[0188] J. Screening for IL-17, IL-18, 4-1BB, CD30 and OX40 Antagonists

[0189] IL-17, IL-18, 4-1BB, CD30 and OX40 antagonists can be evaluatedusing screening assays known in the art, such as high throughput testsystems. The assays can be performed in a variety of formats, includingprotein-protein binding assays, competition binding assays, biochemicalscreening assays, immunoassays, cell based assays, etc. For the sake ofclarity, the following examples describe examplary assays in the contextof IL-17 and IL-17R and are therefore illustrative and not limiting. Thesame assay formats and underlying rationale are equally applicable toIL-18:IL-18R, 4-1BB-L:4-1BB, CD30-L:CD30 and OX40-L:OX40 interactionsfor screening for respective antagonists.

[0190] By observing the effect that an IL-17 antagonist has on theinteraction between IL-17 and IL-17 receptor in various binding assays,on IL-17/IL-17 receptor-mediated activity in functional tests, and incell based screens, molecules that are potential therapeutics areidentified because they inhibit the interaction between IL-17 and IL-17receptor. IL-17 antagonists that partially or completely inhibit IL-17binding to IL-17 receptor, and hence the activation of IL-17 receptor,can be useful as immunosuppressants or anti-inflammatory agents in thetreatment of cardiovascular disease.

[0191] One embodiment of a screening assay that can be used to screenIL-17 antagonists for their ability to inhibit the interaction of IL-17and IL-17 receptor comprises the steps of forming a compositioncomprising an IL-17 protein, an IL-17 receptor protein, and the testcompound (i.e., a putative IL-17 antagonist); assaying for the level ofinteraction of the IL-17 protein, an IL-17 receptor protein; andcomparing the level obtained in the presence of the test compound tothat obtained in the absence of the test compound, such that if thelevel obtained differs, a compound that affects the interaction of IL-17and IL-17 receptor is identified. In alternative embodiments, at leastone of the IL-17 or IL-17 receptor can be labeled with a detectablemoiety. In alternative embodiments, one of the IL-17 or IL-17 receptorcan be soluble, and the other can be bound, although alternative assayformats are possible and well known. The test compound can be added tothe composition after addition of the IL-17 and IL-17 receptor, beforeboth proteins are added, or after one protein is added and before theother is added.

[0192] In another aspect, the screening methods comprise forming acomposition comprising the test compound, the IL-17 protein and cellsexpressing IL-17 receptor; determining the level of biological activityof IL-17 on the IL-17 receptor in the composition; and comparing thelevel of biological activity with that which occurs in the absence oftest compound, wherein a difference in the level of biological activityindicates that the test compound affects the biological activity of theIL-17/IL-17 receptor complex. Biological activity of IL-17 on the IL-17receptor can be assayed in any number of ways, for example but notlimited to, determining the phosphorylation state of intracellularproteins (i.e., activation of the IL-17 receptor by IL-17); determiningthe production of proinflammatory factors, such as IL-6, IL-8, monocytechemoattractant protein-1 and Groα; determining the production ofhematopoietic cytokines, such as G-CSF and GM-CSF and IL-8; anddetermining increased expression of IL-1β and TNF-α, as well asmeasuring induction of iNOS in macrophages.

[0193] A particular example of an assay for the identification ofpotential IL-17 antagonists is a competitive assay, which combines IL-17and an IL-17 receptor-specific antagonist with IL-17 receptor under theappropriate conditions for a competitive assay. Either IL-17 or theIL-17 receptor-specific antagonist can be labeled so that the bindingcan be measured and the effectiveness of the antagonist judged. Thelabel allows for detection by direct or indirect means. Direct meansinclude, but are not limited to luminescence, radioactivity, optical orelectron density. Indirect means include but are not limited to anenzyme or epitope tag.

[0194] Another method by which IL-17 antagonists can be identified thatinhibit the interaction between IL-17 and IL-17 receptor is the solidphase method, in which IL-17 receptor is bound and placed in a mediumwith labeled IL-17. The amount of signal produced by the interactionbetween IL-17 and IL-17 receptor is measured in the presence and in theabsence of a test compound. Diminished levels of signal, in comparisonto a control, indicate that the test compound inhibited the interactionbetween IL-17 and IL-17 receptor. Increased levels of signal, incomparison to a control, indicate that the candidate molecule promotesthe interaction between IL-17 and IL-17 receptor. In alternativeembodiments, IL-17 could be bound and IL-17 receptor labeled. The IL-17antagonist, IL-17 receptor and/or IL-17 proteins can be directly orindirectly labeled. For example, if the protein is recombinantlyproduced, one can engineer fusion proteins that can facilitatesolubility, labeling, immobilization and/or detection. Fusion proteinswhich facilitate these processes can include, but are not limited tosoluble Ig-tailed fusion proteins and His-tagged proteins. Methods forengineering such soluble Ig-tailed fusion proteins are well known tothose of skill in the art. See, for example, U.S. Pat. No. 5,116,964,and the illustrative embodiments described below. Indirect labelinginvolves the use of a protein, such as a labeled antibody, whichspecifically binds to a component of the assay.

[0195] IL-17, IL-18, 4-1BB, CD30 and OX40 antagonists can be identifiedand evaluated using cells and/or cell lines derived from heart andvascular tissues. For example, cardiomyocyte cells and cell lines may beused to evaluate IL-17, IL-18, 4-1BB, CD30 and OX40 antagonists in anyof the suitable assays described herein. Biologically relevant readoutsin the cardiomyocyte-based assay (or other cells) may be used toevaluate potential antagonists, such as cell survival; hypertrophicresponses; and/or production of ANP and/or BNP in response to hypoxic orenvironmental stress.

[0196] IL-17, IL-18, 4-1BB, CD30 and OX40 antagonists can also beidentified using methods that are well suited for high-throughputscreening procedures, such as scintillation proximity assays (Udenfriendet al., 1985, Proc Natl Acad Sci USA 82: 8672-8676), yeast two-hybrid orinteraction trap assays, homogeneous time-resolved fluorescence methods(Park et al., 1999, Anal Biochem 269: 94-104), fluorescence resonanceenergy transfer (FRET) methods (Clegg R M, 1995, Curr Opin Biotechnol 6:103-110), or methods that measure any changes in surface plasmonresonance when a bound polypeptide is exposed to a potential bindingpartner, using for example a biosensor such as that supplied by BiacoreA B (Uppsala, Sweden).

[0197] Compounds that can be assayed that may also be IL-17, IL-18,4-1BB, CD30 and OX40 antagonists include but are not limited to smallorganic molecules, such as those that are commercially available—oftenas part of large combinatorial chemistry compound ‘libraries’—fromcompanies such as Sigma-Aldrich (St. Louis, Mo.), Arqule (Woburn,Mass.), Enzymed (Iowa City, Iowa), Maybridge Chemical Co.(Trevillett,Cornwall, UK), MDS Panlabs (Bothell, Wash.), Pharmacopeia (Princeton,N.J.), and Trega (San Diego, Calif.). Preferred small organic moleculesfor screening using these assays are usually less than 10K molecularweight and can possess a number of physicochemical and pharmacologicalproperties which enhance cell penetration, resist degradation, and/orprolong their physiological half-lives (Gibbs, J., 1994, PharmaceuticalResearch in Molecular Oncology, Cell 79(2): 193-198). Compoundsincluding natural products, inorganic chemicals, and biologically activematerials such as proteins and toxins can also be assayed using thesemethods for the ability to bind to serve as IL-17, IL-18, 4-1BB, CD30and OX40 antagonists.

[0198] Antagonizing IL-17:IL-17R, IL-18:IL-18R, 4-1BB-L:4-1BB,CD30-L:CD30 and/or OX40-L:OX40 interactions and therefore intercellularcommunication, cell stimulation, or immune cell activity can bemanipulated by IL-17, IL-18, 4-1BB, CD30 and OX40 antagonists to controlthese activities in target cells. For example, IL-17, IL-18, 4-1BB, CD30and OX40 antagonists or nucleic acids encoding IL-17, IL-18, 4-1BB, CD30and OX40 antagonists can be administered to a cell or group of cells toblock IL-17:IL-17R, IL-18:IL-18R, 4-1BB-L:4-1BB, CD30-L:CD30 and/orOX40-L:OX40 binding and thereby suppress or arrest cellularcommunication, cell stimulation, or activity in the target cells. Insuch an assay, one would determine a rate of communication or cellstimulation in the presence of the IL-17:IL-17R, IL-18:IL-18R,4-1BB-L:4-1BB, CD30-L:CD30 and/or OX40-L:OX40 binding and then determineif such communication or cell stimulation is altered in the presence ofIL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonists. Exemplary assays forthis aspect of the invention include cytokine secretion assays, T-cellco-stimulation assays, and mixed lymphocyte reactions involving antigenpresenting cells and T cells. These assays are well known to thoseskilled in the art.

[0199] IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonists may regulatecytokine, cell proliferation (either inducing or inhibiting), or celldifferentiation (either inducing or inhibiting) activity, or may induceproduction of other cytokines in certain cell populations. Manypolypeptide factors discovered to date have exhibited such activity inone or more factor-dependent cell proliferation assays, and hence theassays serve as a convenient confirmation of cell stimulatory activity.The activity of IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonists may beevidenced by any one of a number of routine factor-dependent cellproliferation assays for cell lines including, without limitation, NFκB,32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5,DA1, 123, T1165 , HT2, CTLL2, TF-1, Mo7e and CMK.

[0200] The activity of IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonistsmay, among other means, be measured by the following methods:

[0201] Assays for receptor-ligand activity include without limitationthose described in: Current Protocols in Immunology Coligan et al. eds,Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of cellular adhesion under static conditions7.28.1-7.28.22), Takai et al., PNAS USA 84:6864-6868, 1987; Bierer etal., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med.169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al., Cell 80:661-670, 1995.

[0202] Assays for T-cell or thymocyte proliferation include withoutlimitation those described in: Current Protocols in Immunology, Coliganet al. eds, Greene Publishing Associates and Wiley-Interscience (pp.3.1-3.19: In vitro assays for mouse lymphocyte function; Chapter 7:Immunologic studies in humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990;Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli,et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152:1756-1761, 1994.

[0203] Assays for cytokine production and/or proliferation of spleencells, lymph node cells or thymocytes include, without limitation, thosedescribed in: Kruisbeek and Shevach, 1994, Polyclonal T cellstimulation, in Current Protocols in Immunology, Coligan et al. eds. Vol1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto; and Schreiber, 1994,Measurement of mouse and human interferon gamma in Current Protocols inImmunology, Coligan et al. eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley andSons, Toronto.

[0204] Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Bottomly et al., 1991, Measurement of human and murine interleukin 2 andinterleukin 4, in Current Protocols in Immunology, Coligan et al. eds.Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto; deVries et al., JExp Med 173: 1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988;Greenberger et al., Proc Natl Acad Sci.USA 80: 2931-2938, 1983; Nordan,1991, Measurement of mouse and human interleukin 6, in Current Protocolsin Immunology Coligan et al. eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley andSons, Toronto; Smith et al., Proc Natl Acad Sci USA 83: 1857-1861, 1986;Bennett et al., 1991, Measurement of human interleukin 11, in CurrentProtocols in Immunology Coligan et al. eds. Vol 1 pp. 6.15.1 John Wileyand Sons, Toronto; Ciarletta et al., 1991, Measurement of mouse andhuman Interleukin 9, in Current Protocols in Immunology; Coligan et al.eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.

[0205] Assays for T-cell clone responses to antigens (which willidentify, among others, polypeptides that affect APC-T cell interactionsas well as direct T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Coligan et al. eds, Greene PublishingAssociates and Wiley-Interscience (Chapter 3: In vitro assays for mouselymphocyte function; Chapter 6: Cytokines and their cellular receptors;Chapter 7: Immunologic studies in humans); Weinberger et al., PNAS USA77: 6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981;Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988

[0206] Assays for thymocyte or splenocyte cytotoxicity include, withoutlimitation, those described in: Current Protocols in Immunology, Coliganet al. eds, Greene Publishing Associates and Wiley-Interscience (Chapter3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Herrmann et al., PNAS USA 78:2488-2492,1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500,1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., PNASUSA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J.Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998;Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al.,Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.153:3079-3092, 1994.

[0207] Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, polypeptides that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J Immunol144: 3028-3033, 1990; and Mond and Brunswick, 1994, Assays for B cellfunction: in vitro antibody production, in Current Protocols inImmunology Coligan et al. eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley andSons, Toronto.

[0208] Mixed lymphocyte reaction (MLR) assays (which will identify,among others, polypeptides that generate predominantly Th1 and CTLresponses) include, without limitation, those described in: CurrentProtocols in Immunology, Coligan et al. eds, Greene PublishingAssociates and Wiley-Interscience (Chapter 3, In Vitro assays for MouseLymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans);Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

[0209] Dendritic cell-dependent assays (which will identify, amongothers, polypeptides expressed by dendritic cells that activate naiveT-cells) include, without limitation, those described in: Guery et al.,J. Immunol 134:536-544, 1995; Inaba et al., J Exp Med 173:549-559, 1991;Macatonia et al., J Immunol 154:5071-5079, 1995; Porgador et al., J ExpMed 182:255-260, 1995; Nair et al., J Virology 67:4062-4069, 1993; Huanget al., Science 264:961-965, 1994; Macatonia et al., J Exp Med169:1255-1264, 1989; Bhardwaj et al., J Clin Invest 94:797-807, 1994;and Inaba et al., J Exp Med 172:631-640,1990.

[0210] Assays for lymphocyte survival/apoptosis (which will identify,among others, polypeptides that prevent apoptosis after superantigeninduction and polypeptides that regulate lymphocyte homeostasis)include, without limitation, those described in: Darzynkiewicz et al.,Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993;Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell66:233-243, 1991; Zacharchuk, J Immunol 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

[0211] Assays for polypeptides that influence early steps of T-cellcommitment and development include, without limitation, those describedin: Antica et al., Blood 84:111-117, 1994; Fine et al., Cell Immunol155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,PNAS USA 88:7548-7551, 1991

[0212] Assays for embryonic stem cell differentiation (which willidentify, among others, polypeptides that influence embryonicdifferentiation hematopoiesis) include, without limitation, thosedescribed in: Johansson et al. Cellular Biology 15:141-151, 1995; Kelleret al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan etal., Blood 81:2903-2915, 1993.

[0213] Assays for cell movement and adhesion include, withoutlimitation, those described in: Current Protocols in Immunology Coliganet al. eds, Greene Publishing Associates and Wiley-Interscience (Chapter6.12, Measurement of alpha and beta chemokines 6.12.1-6.12.28); Taub etal. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146,1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. JImmunol. 152:5860-5867, 1994; Johnston et al. J Immunol. 153: 1762-1768,1994

[0214] Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419,1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

II. Therapuetic Compositions and Administration thereof

[0215] This invention provides compounds, compositions, and methods fortreating a subject, preferably a human patient, who is suffering fromcardiovascular disease. The terms “treat”, “treating”, and “treatment”used herein includes curative, preventative (e.g., prophylactic) andpalliative or ameliorative treatment. Therapeutic compositions of IL-17,IL-18, 4-1BB, CD30 and/or OX40 antagonist and/or CD39 may therefore needto be administered before, during, or after the presentation ofsymptoms. For therapeutic use, a soluble IL-17, IL-18, 4-1BB, CD30and/or OX40 antagonist and/or CD39 is administered to a subject fortreatment in a manner appropriate to the indication.

[0216] Embodiments of the invention include therapeutic compositions(also referred to as pharmaceutical compositions) comprising one or moresoluble IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonists and/or CD39. A“therapeutic composition,” as used herein, comprises one or more solubleIL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonists and/or CD39 and apharmaceutically acceptable diluent, preservative, solubilizer,emulsifier, adjuvant and/or carrier. As used herein, the terms“pharmaceutically” acceptable and “physiologically” acceptable are usedinterchangeably. The term “pharmaceutically acceptable” means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s).

[0217] Therefore, therapeutic compositions comprise all of theantagonists described in the sections above: e.g., soluble receptormolecules, ligands and/or binding proteins, such as IL-17, IL-17R,IL-18, IL-18R, IL-18 binding protein (IL-18BP), 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L and CD39, as well as biologically active fragments,muteins, variants, derivatives, fusions, etc. thereof; antibodies,fusion proteins and/or peptibodies directed against one or more of thefollowing: IL-17, IL-17R, IL-18, IL-18R, IL-18 binding protein(IL-18BP), 4-1BB, 4-1BB-L, CD30, CD30-L, OX40, OX40-L and CD39; smallmolecules, such as peptidomimetics, mimotopes and the like, thatantagonize the interaction between IL-17 and IL-17R, IL-18 and IL-18R,CD30-L and CD30, 4-1BB-L and 4-1BB and/or OX40-L and OX40; antisenseoligonucleotides that specifically target and hybridize to the mRNA ofendogenous IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30,CD30-L, OX40, OX40-L or CD39 to inhibit or prevent translation of IL-17,IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB, 4-1BB-L, CD30, CD30-L, OX40,OX40-L or CD39 mRNA transcripts; and RNA-interference molecules tailoredto silence expression of IL-17, IL-17R, IL-18, IL-18R, IL-18BP, 4-1BB,4-1BB-L, CD30, CD30-L, OX40, OX40-L or CD39.

[0218] Physiologically acceptable carriers, excipients or diluents arenontoxic to recipients at the dosages and concentrations employed.Ordinarily, preparing such compositions entails combining the IL-17,IL-18, 4-1BB, CD30 and/or OX40 antagonist and/or CD39 with buffers,antioxidants such as ascorbic acid, low molecular weight polypeptides(such as those having fewer than 10 amino acids), proteins, amino acids,carbohydrates such as glucose, sucrose or dextrins, chelating agentssuch as EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with conspecific serum albumin areexemplary appropriate diluents. The IL-17, IL-18, 4-1BB, CD30 and/orOX40 antagonist and/or CD39 preferably is formulated as a lyophilizateusing appropriate excipient solutions (e.g., sucrose) as diluents.Appropriate dosages can be determined in standard dosing trials, and mayvary according to the chosen route of administration. In accordance withappropriate industry standards, preservatives may also be added, such asbenzyl alcohol. The amount and frequency of administration will depend,of course, on such factors as the nature and severity of the indicationbeing treated, the desired response, the age and condition of thepatient, and so forth

[0219] In one embodiment, sustained-release forms of soluble IL-17,IL-18, 4-1BB, CD30 and/or OX40 antagonist and/or CD39 described herein,are used. Sustained-release forms suitable for use in the disclosedmethods include, but are not limited to, IL-17, IL-18, 4-1BB, CD30and/or OX40 antagonist and/or CD39, that is encapsulated in aslowly-dissolving biocompatible polymer, admixed with such a polymer,and or encased in a biocompatible semi-permeable implant. In addition,the IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonist and/or CD39 may beconjugated with polyethylene glycol (pegylated) to prolong its serumhalf-life or to enhance protein delivery (as described in detail above).

[0220] One type of sustained release technology that may be used inadministering soluble IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonistand/or CD39 therapeutic compositions is that utilizing hydrogelmaterials, for example, photopolymerizable hydrogels (Sawhney et al.,Macromolecules 26:581; 1993). Similar hydrogels have been used toprevent postsurgical adhesion formation (Hill-West et al., Obstet.Gynecol. 83:59, 1994) and to prevent thrombosis and vessel narrowingfollowing vascular injury (Hill-West et al., Proc. Natl. Acad. Sci. USA91:5967, 1994). Polypeptides can be incorporated into such hydrogels toprovide sustained, localized release of active agents (West and Hubbel,Reactive Polymers 25:139, 1995; Hill-West et al., J. Surg. Res. 58:759;1995). The sustained, localized release of IL-17, IL-18, 4-1BB, CD30and/or OX40 antagonist and/or CD39 when incorporated into hydrogelswould be amplified by the long half life of IL-17, IL-18, 4-1BB, CD30and/or OX40 antagonist and/or CD39.

[0221] Therapeutic compositions may be for administration for injection,or for oral, pulmonary, nasal, transdermal or other forms ofadministration. In general, the invention encompasses therapeuticcompositions comprising effective amounts one or more IL-17, IL-18,4-1BB, CD30 and/or OX40 antagonist and/or CD39 together withpharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; additives such as detergents and solubilizingagents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol); incorporationof the material into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes.Hyaluronic acid may also be used, and this may have the effect ofpromoting sustained duration in the circulation. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the present proteins and derivatives. See,e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, Pa. 18042) pages 1435-1712 which are hereinincorporated by reference in their entirety. The compositions may beprepared in liquid form, or may be in dried powder, such as lyophilizedform. Implantable sustained release formulations are also contemplated,as are transdermal formulations.

[0222] Contemplated for use herein are oral solid dosage forms, whichare described generally in Chapter 89 of Remington's PharmaceuticalSciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, whichis herein incorporated by reference in its entirety. Solid dosage formsinclude tablets, capsules, pills, troches or lozenges, cachets orpellets. Also, liposomal or proteinoid encapsulation may be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given in Chapter 10of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker andC. T. Rhodes, herein incorporated by reference in its entirety. Ingeneral, the formulation will include one or more IL-17, IL-18, 4-1BB,CD30 and/or OX40 antagonist and/or CD39, and inert ingredients whichallow for protection against the stomach environment, and release of thebiologically active material in the intestine.

[0223] Also specifically contemplated are oral dosage forms of the aboveinventive compounds. If necessary, the compounds may be chemicallymodified delivery is efficacious. Generally, the chemical modificationcontemplated is the attachment of at least one moiety to the compoundmolecule itself, where said moiety permits (a) inhibition ofproteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound and increase in circulation time in the body. Moieties usefulas covalently attached vehicles in this invention may also be used forthis purpose. Examples of such moieties include: PEG, copolymers ofethylene glycol and propylene glycol, carboxymethyl cellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, forexample, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymesas Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, NewYork, N.Y., pp. 367-83; Newmark, et al. (1982), J. Appl. Biochem.4:185-9. Other polymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicatedabove, are PEG moieties. For oral delivery dosage forms, it is alsopossible to use a salt of a modified aliphatic amino acid, such assodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier toenhance absorption of the therapeutic compounds of this invention. Theclinical efficacy of a heparin formulation using SNAC has beendemonstrated in a Phase II trial conducted by Emisphere Technologies.See U.S. Pat. No. 5,792,451, “Oral drug delivery composition andmethods”.

[0224] The compounds of this invention can be included in theformulation as fine multiparticulates in the form of granules or pelletsof particle size about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

[0225] Colorants and flavoring agents may all be included. For example,the protein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

[0226] One may dilute or increase the volume of the compound of theinvention with an inert material. These diluents could includecarbohydrates, especially mannitol, α-lactose, anhydrous lactose,cellulose, sucrose, modified dextrans and starch. Certain inorganicsalts may also be used as fillers including calcium triphosphate,magnesium carbonate and sodium chloride. Some commercially availablediluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0227] Disintegrants may be included in the formulation of thetherapeutic into a solid dosage form. Materials used as disintegrantsinclude but are not limited to starch including the commercialdisintegrant based on starch, Explotab. Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite may all be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

[0228] Binders may be used to hold the therapeutic agent together toform a hard tablet and include materials from natural products such asacacia, tragacanth, starch and gelatin. Others include methyl cellulose(MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

[0229] An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

[0230] Glidants that might improve the flow properties of the drugduring formulation and to aid rearrangement during compression might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

[0231] To aid dissolution of the compound of this invention into theaqueous environment a surfactant might be added as a wetting agent.Surfactants may include anionic detergents such as sodium laurylsulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.Cationic detergents might be used and could include benzalkoniumchloride or benzethonium chloride. The list of potential nonionicdetergents that could be included in the formulation as surfactants arelauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenatedcastor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65and 80, sucrose fatty acid ester, methyl cellulose and carboxymethylcellulose. These surfactants could be present in the formulation of theprotein or derivative either alone or as a mixture in different ratios.

[0232] Additives may also be included in the formulation to enhanceuptake of the compound. Additives potentially having this property arefor instance the fatty acids oleic acid, linoleic acid and linolenicacid.

[0233] Controlled release formulation may be desirable. The compound ofthis invention could be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms; e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compounds of this invention is by a method based on the Orostherapeutic system (Alza Corp.), i.e., the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

[0234] Other coatings may be used for the formulation. These include avariety of sugars which could be applied in a coating pan. Thetherapeutic agent could also be given in a film coated tablet and thematerials used in this instance are divided into 2 groups. The first arethe nonenteric materials and include methyl cellulose, ethyl cellulose,hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropylcellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methylcellulose, providone and the polyethylene glycols. The second groupconsists of the enteric materials that are commonly esters of phthalicacid.

[0235] A mix of materials might be used to provide the optimum filmcoating. Film coating may be carried out in a pan coater or in afluidized bed or by compression coating.

[0236] Also contemplated herein is pulmonary delivery of the presentprotein (or derivatives thereof). The protein (or derivative) isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. (Other reports of thisinclude Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990),Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet etal. (1989), J. Cardiovasc. Phannacol. 13 (suppl.5): s.143-146(endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12((α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6((α1-proteinase); Oswein et al. (March 1990), “Aerosolization ofProteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colorado(recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140:3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

[0237] Contemplated for use in the practice of this invention are a widerange of mechanical devices designed for pulmonary delivery oftherapeutic products, including but not limited to nebulizers, metereddose inhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

[0238] All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

[0239] The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 μm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

[0240] Pharmaceutically acceptable carriers include carbohydrates suchas trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations may include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants may be used. PEG may be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, may be used. Bile salts and otherrelated enhancers may be used. Cellulose and cellulose derivatives maybe used. Amino acids may be used, such as use in a buffer formulation.

[0241] Also, the use of liposomes, microcapsules or microspheres,inclusion complexes, or other types of carriers is contemplated.

[0242] Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

[0243] Formulations for use with a metered-dose inhaler device willgenerally comprise a finely divided powder containing the inventivecompound suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

[0244] Formulations for dispensing from a powder inhaler device willcomprise a finely divided dry powder containing the inventive compoundand may also include a bulking agent, such as lactose, sorbitol,sucrose, mannitol, trehalose, or xylitol in amounts which facilitatedispersal of the powder from the device, e.g., 50 to 90% by weight ofthe formulation.

[0245] Nasal delivery of IL-17, IL-18, 4-1BB, CD30 and/or OX40antagonist and/or CD39 is also contemplated. Nasal delivery allows thepassage of the protein to the blood stream directly after administeringthe therapeutic product to the nose, without the necessity fordeposition of the product in the lung. Formulations for nasal deliveryinclude those with dextran or cyclodextran. Delivery via transportacross other mucous membranes is also contemplated.

[0246] In practicing the method of treatment or use of IL-17, IL-18,4-1BB, CD30 and/or OX40 antagonist and/or CD39, a therapeuticallyeffective amount is administered to a subject. As used herein, the term“therapeutically effective amount” means the total amount of eachtherapeutic composition that is sufficient to show a meaningful patientbenefit, i.e., treatment, healing, prevention or amelioration of therelevant medical condition, or an increase in rate of treatment,healing, prevention or amelioration of such conditions. When applied toan individual therapeutic composition, administered alone, the termrefers to that ingredient alone. When applied to a combination, the termrefers to combined amounts of the ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. As used herein, the phrase “administering atherapeutically effective amount” of a therapeutic agent means that thepatient is treated with said therapeutic composition in an amount andfor a time sufficient to induce an improvement, and preferably asustained improvement, in at least one indicator that reflects theseverity of the disorder. An improvement is considered “sustained” ifthe patient exhibits the improvement on at least two occasions separatedby one or more days, or more preferably, by one or more weeks. Thedegree of improvement is determined based on signs or symptoms, anddeterminations can also employ questionnaires that are administered tothe patient, such as quality-of-life questionnaires. Various indicatorsthat reflect the extent of the patient's illness can be assessed fordetermining whether the amount and time of the treatment is sufficient.The baseline value for the chosen indicator or indicators is establishedby examination of the patient prior to administration of the first doseof the therapeutic agent. Preferably, the baseline examination is donewithin about 60 days of administering the first dose. If the therapeuticagent is being administered to treat acute symptoms, the first dose isadministered as soon as practically possible after the injury hasoccurred. Improvement is induced by administering therapeuticcompositions such as IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonistand/or CD39 until the patient manifests an improvement over baseline forthe chosen indicator or indicators. In treating chronic conditions, thisdegree of improvement is obtained by repeatedly administering thismedicament over a period of at least a month or more, e.g., for one,two, or three months or longer, or indefinitely. A period of one to sixweeks, or even a single dose, often is sufficient for treating injuriesor other acute conditions. Although the extent of the patient's illnessafter treatment may appear improved according to one or more indicators,treatment may be continued indefinitely at the same level or at areduced dose or frequency. Once treatment has been reduced ordiscontinued, it later may be resumed at the original level if symptomsshould reappear.

[0247] One skilled in the pertinent art will recognize that suitabledosages will vary, depending upon such factors as the nature andseverity of the disorder to be treated, the patient's body weight, age,general condition, and prior illnesses and/or treatments, and the routeof administration. Preliminary doses can be determined according toanimal tests, and the scaling of dosages for human administration isperformed according to art-accepted practices such as standard dosingtrials. For example, the therapeutically effective dose can be estimatedinitially from cell culture assays. The dosage will depend on thespecific activity of the compound and can be readily determined byroutine experimentation. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture,while minimizing toxicities. Such information can be used to moreaccurately determine useful doses in humans. Ultimately, the attendingphysician will decide the amount of polypeptide of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of polypeptide of the presentinvention and observe the patient's response. Larger doses ofpolypeptide of the present invention can be administered until theoptimal therapeutic effect is obtained for the patient, and at thatpoint the dosage is not increased further. It is contemplated that thevarious therapeutic compositions used to practice the method of thepresent invention should contain about 0.01 ng to about 100 mg(preferably about 0.1 ng to about 10 mg, more preferably about 0.1microgram to about 1 mg) of polypeptide of the present invention per kgbody weight. In one embodiment of the invention, IL-17, IL-18, 4-1BB,CD30 and/or OX40 antagonist and/or CD39 are administered one time perweek to treat the various medical disorders disclosed herein, in anotherembodiment is administered at least two times per week, and in anotherembodiment is administered at least three times per week. If injected,the effective amount of IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonistand/or CD39 per adult dose ranges from 1-20 mg/m², and preferably isabout 5-12 mg/m². Alternatively, a flat dose can be administered, whoseamount may range from 5-100 mg/dose. Exemplary dose ranges for a flatdose to be administered by subcutaneous injection are 5-25 mg/dose,25-50 mg/dose and 50-100 mg/dose. In one embodiment of the invention,the various indications described below are treated by administering apreparation acceptable for injection containing IL-17, IL-18, 4-1BB,CD30 and/or OX40 antagonist and/or CD39 at 25 mg/dose, or alternatively,containing 50 mg per dose. The 25 mg or 50 mg dose can be administeredrepeatedly, particularly for chronic conditions. If a route ofadministration other than injection is used, the dose is appropriatelyadjusted in accord with standard medical practices. In many instances,an improvement in a patient's condition will be obtained by injecting adose of about 25 mg of IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonistand/or CD39 one to three times per week over a period of at least threeweeks, or a dose of 50 mg of IL-17, IL-18, 4-1BB, CD30 and/or OX40antagonist and/or CD39 one or two times per week for at least threeweeks, though treatment for longer periods may be necessary to inducethe desired degree of improvement. For incurable chronic conditions, theregimen can be continued indefinitely, with adjustments being made todose and frequency if such are deemed necessary by the patient'sphysician. The foregoing doses are examples for an adult patient who isa person who is 18 years of age or older. For pediatric patients (age4-17), a suitable regimen involves the subcutaneous injection of 0.4mg/kg, up to a maximum dose of 25 mg of IL-17, IL-18, 4-1BB, CD30 and/orOX40 antagonist and/or CD39, administered by subcutaneous injection oneor more times per week. If an IL-17, IL-18, 4-1BB, CD30 and/or OX40antagonist is in the form of an antibody, a preferred dose range is 0.1to 20 mg/kg, and more preferably is 1-10 mg/kg. Another embodiment of adose range is 0.75 to 7.5 mg/kg of body weight. Humanized antibodies arepreferred, that is, antibodies in which only the antigen-binding portionof the antibody molecule is derived from a non-human source. Suchantibodies can be injected or administered intravenously.

III. Therapeutic Applications

[0248] IL-17, IL-18, 4-1BB, CD30 and OX40 antagonists may be used totreat cardiovascular disease. Embodiments of the present inventioninclude methods of treating cardiovascular disease in a subject havinghaving cardiovascular disease comprising administering an effectiveamount of one or more IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonistsand/or CD39, alone or in any combination.

[0249] Cardiovascular disease includes disease states havingpathophysiology of the heart and vasculature systems, as well as organsand systems compromised by disease states of the heart and vasculaturesystems. Examples include, but are not limited to: inflammation of theheart and/or vasculature such as myocarditis, chronic autoimmunemyocarditis, bacterial and viral myocarditis, as well as infectiveendocarditis; heart failure; congestive heart failure; chronic heartfailure; cachexia of heart failure; cardiomyopathy, includingnon-ischemic (dilated cardiomyopathy; idiopathic dilated cardiomyopathy;cardiogenic shock, heart failure secondary to extracorporeal circulatorysupport (“post-pump syndrome”), heart failure followingischemia/reperfusion injury, brain death associated heart failure (asdescribed in Owen et al., 1999 (Circulation. 1999 May18;99(19):2565-70)); hypertrophic cardiomyopathy; restrictivecardiomyopathy; non-ischemic systemic hypertension; valvular disease;arythmogenic right ventricular cardiomyopathy) and ischemic(atherogenesis; atherosclerosis; arteriosclerosis; peripheral vasculardisease; coronary artery disease; infarctions, including stroke,transient ischemic attacks and myocardial infarctions). Additionaldisease states encompassed by the definition of cardiovascular diseaseinclude: aneurysms; arteritis; angina; embolism; platelet-associatedischemic disorders; ischemia/reperfusion injury; restenosis; mitraland/or tricuspid regurgitation; mitral stenosis; silent myocardialischemia; Raynaud's phenomena; thrombosis; deep venous thrombosis;pulmonary embolism; thrombotic microangiopathies including thromboticthrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS),essential thrombocythemia, disseminated intravascular coagulation (DIC),and thrombosis and coagulopathies associated with exposure to a foreignor injured tissue surfacethrombophlebitis; vasculitis, includingKawasaki's vasculitis; Takayasu's arteritis; veno-occlusive disease,giant cell arteritis, Wegener's granulomatosis; Schoenlein-Henochpurpura, as well as cardiovascular disease arising from periodontalinfections by one or more oral pathogens, such as bacteria.

[0250] Additional examples of the therapeutic uses of one or more IL-17,IL-18, 4-1BB, CD30 and/or OX40 antagonists alone or in combination withCD39 include the treatment of individuals who suffer from coronaryartery disease or injury following platelet-associated ischemicdisorders including lung ischemia, coronary ischemia, and cerebralischemia, and for the prevention of reocclusion following thrombosis,thrombotic disorders including coronary artery thrombosis, cerebralartery thrombosis, intracardiac thrombosis, peripheral arterythrombosis, venous thrombosis, thrombotic microangiopathies includingthrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome(HUS), essential thrombocythemia, disseminated intravascular coagulation(DIC), and thrombosis and coagulopathies associated with exposure to aforeign or injured tissue surface, in combination with angioplasty,carotid endarterectomy, anastomosis of vascular grafts, and chroniccardiovascular devices such as in-dwelling catheters or shunts.

[0251] Further indications include subjects that are or will beundergoing angioplasty procedures (i.e., balloon angioplasty, laserangioplasty, coronary atherectomy and similar techniques), placement ofendovascular prosthetic devices such as carotid, coronary, peripheralarterial or other endovascular stents, dialysis access devices, orprocedures to treat peripheral vascular disease; individuals undergoingsurgery that has a high risk of thrombus formation (i.e., coronarybypass surgery, insertion of a prosthetic valve or vessel and the like).

[0252] In addition, IL-17 and/or IL-18 are prognostic indicators ofcardiovascular disease and disease severity. IL-17 and/or IL-18 are alsoprognostic indicators of donor adequacy and post-transplant outcome.Therefore, further embodiments of the invention include assays formeasuring IL-17 and/or IL-18 levels in subjects being screened forcardiovascular disease, cardiovascular disease severity, donor adequacyand post-transplant outcome.

[0253] Various indicators that reflect the extent of the patient'sillness may be assessed for determining whether the amount and time ofthe treatment is sufficient. The baseline value for the chosen indicatoror indicators is established by examination of the patient prior toadministration of the first dose of IL-17, IL-18, 4-1BB, CD30 and/orOX40 antagonists alone or in combination with soluble CD39. Preferably,the baseline examination is done within about 60 days of administeringthe first dose.

[0254] Improvement is induced by repeatedly administering a dose ofIL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonists alone or incombination with soluble CD39 until the patient manifests an improvementover baseline for the chosen indicator or indicators. In treatingchronic conditions, this degree of improvement is obtained by repeatedlyadministering this medicament over a period of at least a month or more,e.g., for one, two, or three months or longer, or indefinitely. A periodof one to six weeks, or even a single dose, often is sufficient fortreating acute conditions.

[0255] Although the extent of the patient's illness after treatment mayappear improved according to one or more indicators, treatment may becontinued indefinitely at the same level or at a reduced dose orfrequency. Once treatment has been reduced or discontinued, it later maybe resumed at the original level if symptoms should reappear.

[0256] Therapeutic compositions of the invention may be administeredalone or in combination with a therapeutically effective amount of otherdrugs. The invention includes the administration of IL-17, IL-18, 4-1BB,CD30 and/or OX40 antagonists alone or in combination with soluble CD39concurrently with one or more other drugs that are administered to thesame patient in combination with the IL-17, IL-18, 4-1BB, CD30 and/orOX40 antagonists and/or CD39, each drug being administered according toa regimen suitable for that medicament. “Concurrent administration”encompasses simultaneous or sequential treatment with the components ofthe combination, as well as regimens in which the drugs are alternated,or wherein one component is administered long-term and the other(s) areadministered intermittently. Components can be administered in the sameor in separate compositions, and by the same or different routes ofadministration.

[0257] Examples of other drugs or therapeutic compostions that may beused in combination with IL-17, IL-18, 4-1BB, CD30 and/or OX40antagonists alone or in combination with soluble CD39 include: analgesicagents, disease-modifying anti-rheumatic drugs (DMARDs), non-steroidalanti-inflammatory drugs (NSAIDs), and any immune and/or inflammatorymodulators. Non-steroidal anti-inflammatories may include, but are notlimited to: salicylic acid (aspirin); ibuprofen; indomethacin;celecoxib; rofecoxib; ketorolac; nambumetone; piroxicam; naproxen;oxaprozin; sulindac; ketoprofen; diclofenac; other COX-1 and/or COX-2inhibitors, salicylic acid derivatives, propionic acid derivatives,acetic acid derivatives, fumaric acid derivatives, carboxylic acidderivatives, butyric acid derivatives, oxicams, pyrazoles andpyrazolones, including newly developed anti-inflammatories.

[0258] Therapeutic compositions of this invention may be administeredwith one or more of the following: modulators of other members of theTNF/TNF receptor family, including TNF antagonists, such as etanercept(Enbrel™), sTNF-RI, onercept, D2E7, and Remicade™; IL-1 inhibitors,including IL-1ra molecules such as anakinra and more recently discoveredIL-1ra-like molecules such as IL-1Hy1 and IL-1Hy2; IL-1 “trap” moleculesas described in U.S. Pat. No. 5,844,099; IL-1 antibodies; solubilizedIL-1 receptor, and the like; IL-6 inhibitors (e.g., antibodies to IL-6);IL-8 inhibitors (e.g., antibodies to IL-8); Interleukin-1 convertingenzyme (ICE) modulators; insulin-like growth factors (IGF-1, IGF-2) andmodulators thereof; transforming growth factor-β (TGF-β), TGF-β familymembers, and TGF-β modulators; fibroblast growth factors FGF-1 toFGF-10, and FGF modulators; COX-2 inhibitors, such as Celebrex™ andVioxx™; prostaglandin analogs (e.g., E series prostaglandins); matrixmetalloproteinase (MMP) modulators; nitric oxide synthase (NOS)modulators, including modulators of inducible NOS; modulators ofglucocorticoid receptor; modulators of glutamate receptor; modulators oflipopolysaccharide (LPS) levels; anti-cancer agents, includinginhibitors of oncogenes (e.g., fos, jun) and interferons; noradrenalineand modulators and mimetics thereof.

[0259] Additional embodiments of compositions that can be administeredconcurrently with the pharmaceutical compositions of the invention are:cytokines, lymphokines, or other hematopoietic factors such as M-CSF,GM-CSF, Flt3-Ligand, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0 , TNF1, TNF2,G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin, orinhibitors or antagonists of any of these factors. The pharmaceuticalcomposition can further contain other agents which either enhance theactivity of the polypeptide or compliment its activity or use intreatment. Such additional factors and/or agents may be included in thepharmaceutical composition to produce a synergistic effect withpolypeptide of the invention, or to minimize side effects. Conversely,IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonists and/or soluble CD39may be included in formulations of the particular cytokine, lymphokine,other hematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent to minimize side effects of the cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent.

[0260] Further embodiments of drugs to be administered concurrentlyinclude but are not limited to antivirals, antibiotics, analgesics,corticosteroids, antagonists of inflammatory cytokines, non-steroidalanti-inflammatories, pentoxifylline, thalidomide, and disease-modifyingantirheumatic drugs (DMARDs) such as azathioprine, cyclophosphamide,cyclosporine, hydroxychloroquine sulfate, methotrexate, leflunomide,minocycline, penicillamine, sulfasalazine and gold compounds such asoral gold, gold sodium thiomalate, and aurothioglucose.

[0261] Of course, IL-17, IL-18, 4-1BB, CD30 and/or OX40 antagonistsand/or soluble CD39, as well as other therapeutic compostions describedabove, may be administered inconjuction with other recognized therapiesor treatments, such as any surgical procedures involving the heart andvaculature (coronary bypass, heart transplant, valve replacement,angioplasty, stenting, atherectomy, aortic aneurysm repair, valveplication, ventricular assist device insertion, ventricular volumereduction surgery, any form of peripheral arterial surgery includingbypass, vessel recanalisation or reconstruction, pediatriccardiovascular surgery including repair and correction of complexcongenital lesions); Lipid-lowering drugs (such as, but not limited toLipitor, simvastatin, pravastatin, atorvastatin, non-HMG CoA reductaseinhibitors); blood pressure-regulating drugs (including but not limitedto calcium channel antagonists, ACE-inthibitors, beta-blockers, orallyand systemically available nitric oxide donors such as GTN);angiotensin-converting enzyme inhibitors, and peroxisomeproliferator-activated receptor ligands.

[0262] In other embodiments of the invention, 4-1BB, CD30 and/or OX40antagonists may be used to prevent, reduce and/or ameliorate thecardiotoxicity of chemotherapeutics. Drug toxicity remains a significantbarrier to the delivery of curative doses of cancer chemotherapy. Manychemotherapeutic drugs cause direct injury to the heart, either acutelyin the form of myocardial tissue injury or dysrhythmias, or in a chronicfashion associated with congestive heart failure. Examples of acutecardiotoxicity include supraventricular tachyarrhymias, which may beassociated ECG changes, such as ST-T sgement changes, decreased voltage,T-wave flattening, as well as atrial and ventricular ectopy. Acuteeffects occur in up to 40% of patients receiving bolus doxorubicin andare usually transient. Chronic anthracycline cardiotoxicity may bemanifested as arrythmias, myocarditis, pericarditis, myocardialinfarction and cardiomyopathy that is dose- and schedule-dependent.Above cumulative bolus doses of 550 mg/m² the risk of congestive heartfailure increases rapidly. Doses of less than 450 mg/m² pose a risk ofless than 10%. Patients receiving anthracyclines also demontratelate-appearing cardiac toxicity occuring greater than 5 years afterexposure to doxorubucin. Cardiac dysfunction is manifested as congestiveheart failure or dysrhythmias and can occur in patients that werepreviously asymptomatic. It is estimated that approximtely 5% ofpatients surviving ten years after exposre to doxorubicin willexperience this toxicity (see, Page, R., Cancer Management: AMultidisciplinary Approach, PRR Inc., Fifth Edition (2001).

[0263] Principal among the cardiotoxic agents are cytostatic antibioticsof the anthracycline class. The class of anthracylines includes, but isnot limited to, Adriamycin (Doxorubicin), Daunorubicin, Ellence(Epirubicin), Idarubicin, Mitroxantrone, and the like. Therefore,embodiments of the invention provide methods of preventing, reducingand/or ameliorating the cardiotoxic effects of anthracylines comprisingadministrating an effective amount of a 4-1BB antagonists, CD30antagonists and/or OX40 antagonists. Embodiments of the invention alsoprovide compositions for preventing, reducing and/or ameliorating thecardiotoxic effects of anthracylines comprising an antagonist selectedfrom the group consisting of a 4-1BB antagonists, CD30 antagonistsand/or OX40 antagonists.

[0264] Embodiments of the invention provide methods of preventing,reducing and/or ameliorating the cardiotoxic effects of anthracylinesselected from the group consisting of Adriamycin (Doxorubicin),Daunorubicin, Ellence (Epirubicin), Idarubicin and Mitroxantrone,comprising administrating an effective amount of a 4-1BB antagonists,CD30 antagonists and/or OX40 antagonists. Embodiments of the inventionalso provide compositions for preventing, reducing and/or amelioratingthe cardiotoxic effects of anthracylines selected from the groupconsisting of Adriamycin (Doxorubicin), Daunorubicin, Ellence(Epirubicin), Idarubicin and Mitroxantrone, comprising an antagonistselected from the group consisting of a 4-1BB antagonists, CD30antagonists and/or OX40 antagonists.

[0265] 4-1BB antagonists, CD30 antagonists and/or OX40 antagonists maybe used to prevent, reduce and/or ameliorate the cardiotoxic effects ofother chemotherapeutics having cardiotoxicity, such as, but not limitedto: Amsacrine, Busulfan, Cisplatin, Cyclophosphamide, Fluorouracil,Herceptin (and other Her2/neu-targeted modalities), Ifosfamide,Interferons, Interleukin-2, Mitomycin, Paclitaxel, Vinblastine,Vincristine and Xeloda (capecitabine).

[0266] Therefore, embodiments of the invention provide methods ofpreventing, reducing and/or ameliorating the cardiotoxic effects ofchemotherapeutics having cardiotoxic side effects, comprisingadministrating an effective amount of a 4-1BB antagonists, CD30antagonists and/or OX40 antagonists. Embodiments of the invention alsoprovide compositions for preventing, reducing and/or ameliorating thecardiotoxic effects of chemotherapeutics having cardiotoxic side effectscomprising an antagonist selected from the group consisting of a 4-1BBantagonists, CD30 antagonists and/or OX40 antagonists.

[0267] Embodiments of the invention provide methods of preventing,reducing and/or ameliorating the cardiotoxic effects ofchemotherapeutics selected from the group consisting of Amsacrine,Busulfan, Cisplatin, Cyclophosphamide, Fluorouracil, Herceptin (andother Her2/neu-targeted modalities), Ifosfamide, Interferons,Interleukin-2, Mitomycin, Paclitaxel, Vinblastine, Vincristine andXeloda (capecitabine), comprising administrating an effective amount ofa 4-1BB antagonists, CD30 antagonists and/or OX40 antagonists.Embodiments of the invention also provide compositions for preventing,reducing and/or ameliorating the cardiotoxic effects ofchemotherapeutics selected from the group consisting of Amsacrine,Busulfan, Cisplatin, Cyclophosphamide, Fluorouracil, Herceptin (andother Her2/neu-targeted modalities), Ifosfamide, Interferons,Interleukin-2, Mitomycin, Paclitaxel, Vinblastine, Vincristine andXeloda (capecitabine), comprising an antagonist selected from the groupconsisting of a 4-1BB antagonists, CD30 antagonists and/or OX40antagonists.

[0268] Embodiments of the invention provide methods of treating cancerin a subject in need thereof, wherein the dosage of a chemotherapeutichaving cardiotoxicity is increased to more effectively treat the cancerbut the cardiotoxic effects of the chemotherapeutic is prevented,reduced and/or ameiorated by administering an antagonist selected fromthe group consisting of a 4-1BB antagonists, CD30 antagonists and/orOX40 antagonists. Embodiments of the invention provide methods oftreating cancer in a subject in need thereof, wherein the dosage of ananthracyline is increased to more effectively treat the cancer but thecardiotoxic effects of the anthracycline is prevented, reduced and/orameiorated by administering an antagonist selected from the groupconsisting of a 4-1BB antagonists, CD30 antagonists and/or OX40antagonists. Embodiments of the invention provide methods of treatingcancer in a subject in need thereof, wherein the dosage of ananthracyline selected from the group consisting of Adriamycin(Doxorubicin), Daunorubicin, Ellence (Epirubicin), Idarubicin andMitroxantrone is increased to more effectively treat the cancer but thecardiotoxic effects of the anthracycline is prevented, reduced and/orameiorated by administering an antagonist selected from the groupconsisting of a 4-1BB antagonists, CD30 antagonists and/or OX40antagonists. Embodiments of the invention provide methods of treatingcancer in a subject in need thereof, wherein the dosage of achemotherapeutic selected from the group consisting of Amascrine,Busulfan, Cisplatin, Cyclophosphamide, Fluorouracil, Herceptin (andother Her2/neu-targeted modalities), Ifosfamide, Interferons,Interleukin-2, Mitomycin, Paclitaxel, Vinblastine, Vincristine andXeloda (capecitabine) is increased to more effectively treat the cancerbut the cardiotoxic effects of the chemotherapeutic is prevented,reduced and/or ameiorated by administering an antagonist selected fromthe group consisting of a 4-1BB antagonists, CD30 antagonists and/orOX40 antagonists.

[0269] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0270] The invention having been described, the following examples areoffered by way of illustration, and not limitation. Sequence IdentityNumbers and Associated Molecules SEQ ID NO. Molecule 1 IL-17polynucleotide sequence 2 IL-17 amino acid sequence 3 IL-17 Receptorpolynucleotide sequence 4 IL-17 Receptor amino acid sequence 5 IL-18Receptor: IL-1Rrp1 polynucleotide sequence 6 IL-18 Receptor: IL-1Rrp1amino acid sequence 7 IL-18 Receptor: AcPL polynucleotide sequence 8IL-18 Receptor: AcPL amino acid sequence 9 IL-18 Binding Protein apolynucleotide sequence 10 IL-18 Binding Protein a amino acid sequence11 IL-18 Binding Protein-Fc fusion amino acid sequence 12 IL-18polynucleotide sequence (unprocessed) 13 IL-18 amino acid sequence(unprocessed) 14 IL-18 amino acid sequence (ICE-processed) 15 4-1BB-Lpolynucleotide sequence 16 4-1BB-L amino acid sequence 17 4-1BBpolynucleotide sequence 18 4-1BB amino acid sequence 19 CD30-Lpolynucleotide sequence (nt 1-648) 20 CD30-L polypeptide sequence (aa1-215) 21 CD30-L polynucleotide sequence (nt 1-705) 22 CD30-Lpolypeptide sequence (aa 1-234) 23 CD30 polynucleotide sequence 24 CD30polypeptide sequence 25 OX40-L polynucleotide sequence 26 OX40-Lpolypeptide sequence 27 OX40 polynucleotide sequence 28 OX40 polypeptidesequence 29 CD39 polynucleotide sequence 30 CD39 polypeptide sequence 31Flag ® octapeptide 32 Linker - (Gly)₄Ser(Gly)₅Ser 33 Linker -GlyAlaGlyGlyAlaGlySer(Gly)₅Ser 34 Linker - (Gly₄Ser)₂ 35 Linker -(GlyThrPro)₃ 36 Linker - (Gly₄Ser)₃Gly₄SerGly₅Ser

EXAMPLES Example 1 IL-17 and IL-18 Plasma Levels are Elevated inCardiomyopathy Patients

[0271] These studies demonstrate IL-17 and IL-18 are elevated in humanpatients having various forms and severity of cardiovascular disease.

[0272] In a series of studies, plasma levels of IL-17 and IL-18 werefound to be elevated in patients having acute and chronic heart failure.Plasma from brain-dead organ donors was obtained at the time of heartremoval and stored. The clinical outcome of the recipient that receivedthe various organs was noted. A group of recipients that survived anddid well was collected along with a group that died within 72 hours oftransplantation due to cardiac failure refractory to maximal medicalsupport. The plasma from the original donors, recipients and unuseddonors having an ejection fraction (EF) of less than 30% was assayed forIL-17 and IL-18. Cytokine levels were measured in plasma samplesessentially as described in the protocols provided in commerciallyavailable ELISA kits (see, for example, QUANTKINE® R&D Systems,Minneapolis, Minn., which provides assays for the quantitativedetermination of human IL-17 and human IL-18).

[0273] As shown in FIGS. 1A and 1B, IL-17 and IL-18 were elevated inpatients that died shortly after transplantation in contrast to patientsthat survived. IL-17 and IL-18 were also elevated in unused donorshaving an ejection fraction (EF) of less than 30%, suggesting acorrelation between circulating IL-17 and IL-18 levels and diseaseseverity. This study further shows the diagnostic and prognostic valueof assaying for IL-17 and IL-18 cytokine levels in heart patients, suchas for the assessment of post-transplant survival.

[0274] Using the samples described above, IL-18 receptor expression wasassessed. Heart samples were homogenized in ice-cold lysis buffer (NewEngland Biolabs, Beverly, Mass.). The homogenate was centrifuged at 4°C. (12000×5 mins) and the supernatant assayed for protein content(Pierce BCA kit). The same total amount of protein (20 mg/lane) wassubjected to SDS-PAGE using 5% gels. Proteins were then transferred tonitrocellulose membrane and IL-18 receptor visualized using a polyclonalantibody raised against IL-18R alpha (AF840-R&D Systems, Mineapolis,Minn.), and the ECL kit.

[0275] As shown in FIG. 2 the relative abundance of IL-18 receptor ishigher in patients having an ejection fraction (EF) of less than 30% ascompared to patients in end stage heart failure-ESF (i.e., NYHA stage 4cardiomyopathy) and patients havng an ejection fraction of greater than60%. These data demonstrate that elevated IL-18 receptor expression isassociated with impaired myocardial function. Given that IL-18 signalsthrough the Toll-IL-1 receptor pathway (TIR) and that bothlipopolysacharide and IL-1 are negatively inotropic, elevated expressionof the IL-18 receptor could well account for part of the myocardialdysfunction seen in these patients.

[0276] Samples from cardiac patients participating in the multi-centerRenaissance Trial were evaluated. The patients exhibited a continuim offunctional capacity and objective evidence of cardiovascular disease, asclassifed by the Criteria Committee of the New York Heart Association(Nomenclature and Criteria for Diagnosis of Diseases of the Heart andGreat Vessels. 9^(th) ed. Boston Mass: Little, Brown & co;1994:253-256). Samples were evaluated for cytokine levels using theassays described above.

[0277] FIGS. 3A-3D depict the plasma concentration of IL-17 and IL-18(pg/ml) in cardiomypoathy patients diagnosed in NYHA classes 1, 2, 3a,3b and 4. This data shows a dramatic and unexpected increase in theamount of circulating IL-17 in NYHA classes 2, 3a and 3b (FIG. 1A).Elevated IL-17 levels were found in both non-ischemic and ischemiccardiomyopathy for the same classes (FIG. 1B). IL-17 levels weresignificantly higher in NYHA class 3a for ischemic cardiomyopathy.Significantly, this data shows a direct correlation between IL-17 levelsand progression of disease up to NYHA class 3b and a decrease in IL-17in NYHA class 4.

[0278] Plasma levels of IL-18 were also elevated in NYHA classes 2, 3a,3b and 4 (FIG. 3C). Similar to IL-17, this data shows a directcorrelation between IL-18 levels and progression of disease up to NYHAclass 3b with a decrease in IL-18 in NYHA class 4 (FIG. 3C). When brokendown into non-ischemic and ischemic cardiomyopathy, IL-18 levels wereelevated in both non-ischemic and ischemic cardiomyopathy for the sameNYHA classes (FIG. 3D). Non-ischemic cardiomyopathy patients hadcomparatively higher levels of IL-18 for NYHA classes 2, 3a and 3b.

[0279] This data demonstrates that IL-17 plasma levels can be used as aprognostic indicator of cardiovascular disease and disease severity.Without being bound by theory, the relative expression of IL-17 andIL-18 may be diagnostic of non-ischemic versus ischemic cardiomyopathy(compare FIGS. 3B and 3D). Therefore, assays to detect circulatinglevels of IL-17 and IL-18 may be used to diagnose cardiovascular diseaseand qualitatively assess disease severity. Taken together, this datashows IL-17 and IL-18 are implicated in cardiovascular disease andprovides a basis for treating cardiovascular disease by administeringIL-17 and/or IL-18 antagonists, alone or in combination.

Example 2 Cytokine Profiles of Heart Transplant Recipients

[0280] This study shows, inter alia, that IL-17, IL-18 and soluble 4-1BBare elevated in heart transplant recipients that died within 72 hours oftransplantation. This data shows that myocardial dysfunction isassociated with significantly elevated levels of IL-17, IL-18 andsoluble 4-1BB.

[0281] Cardiac transplantation remains a major therapeutic modality forpatients with end-stage heart failure; however, the number of donororgans significantly limits its availability. This situation is worsenedby the fact that around 20% of organ donors have such severe acutemyocardial dysfunction associated with brain death that their hearts areunable to be used for transplantation (Hosenpud J D, et al., Heart LungTransplant. 2001, 20(8):805-15). Brain death is a catastrophic eventassociated with marked activation of the immune system and elevatedplasma levels of cytokines such as TNFα and IL6 (Takada, M, et al.,Transplantation 1998, 65(12):1533-42 and Birks, E J, et al., TransplantProc. 2001;33(5):2749-51). Cytokines are critical regulators of theT-helper 1 (Th1) and Th2 T-cell responses (Neurath, M F, et al., NatMed. 2002, 8(6):567-73). The Th1 response results in pro-inflammatorycytokine release characterized by macrophage activation and, ifunopposed, may result in tissue damage. The Th2 response results in ahumoral immune response that in general opposes the Th1 response.

[0282] We hypothesized that death early after heart transplantationcould be due to donor derived factors impacting on heart function eitherdirectly or indirectly, for example by initiating an acute rejectionepisode, ultimately resulting in myocardial dysfunction. To test thishypothesis we obtained plasma from two groups of heart transplant donors(as described above). Group A comprised samples from 16 organ donorswhere the recipient had an uneventful postoperative course and survivedgreater than 1 year. Group B samples were obtained from 14 donors wherethe recipients died within 72 hours of transplantation with myocardialdysfunction refractory to maximal medical therapy. The circulatinglevels of the Th1 (pro-inflammatory) cytokines interferon gamma (IFNγ),IL-12, IL-15, IL-17 and IL-18 along with the Th2 (anti-inflammatory)cytokines IL-4, IL-5, IL-10 and IL-13 were measured. Cytokine levelswere determined using LUNIMEX® technology (Upstate, Waltham, Mass.),QUANTIKINE® ELISA kits (R&D Systems) or a custom made ELISA for soluble4-1BB (using capture and detection antibodies, also from R&D systems).The control group consisted of 21 healthy patients withechocardiographically normal myocardial function undergoing routinecoronary artery bypass graft surgery. The circulating Th2/Th1 ratio wasobtained by dividing the sum of the Th2 cytokines by the sum of Th1cytokines, both in pg/ml. In addition, as a potential marker of T-cellinteraction with antigen presenting cells, we also measured the level ofthe soluble receptor 4-1BB. 4-1BB is present on activated T-cells and4-1BB ligand (4-1BB-L) is present on antigen presenting cells;engagement of 4-1BB by 4-1BB-L acts as a co-stimulatory signal (Kwon,B., et al., Mol Cells. 2000;10(2):119-26). Inflammatory mediator levelsin groups A and B were compared to those in 21 non-brain dead controlsubjects.

[0283]FIG. 9 shows that 4-1BB levels were significantly elevated in thepatients with heart failure compared to normal subjects suggestingactivation of this system in human heart failure, in common withrheumatoid arthritis patients (Eur J Immunol. 1998 January;28(1):290-5).

[0284] As shown in the Table 2, plasma samples from brain dead organdonors, where the recipients had an uneventful outcomepost-transplantation (group A), contained significantly elevated levelsof the Th2 cytokines IL-4, 10 and 13, along with the Th1 cytokines IFNγ,IL-12, 17 and 18 compared to control subjects. Overall, however, thismaintained a circulating Th2/Th1 ratio of 0.32, which was notsignificantly different from 0.45 seen in control subjects. In group B,where the recipients died within 72 hours of transplantation, the Th2cytokines IL-4 and 13 were unchanged from control levels, IL-10 waselevated compared to control subjects, but significantly reducedcompared to Group A samples. IFNγ was elevated compared to controlsamples and IL-12 was unaltered. However, marked elevations in thelevels of IL-17 and IL-18 were identified, resulting in a reduction inthe Th2/Th1 ratio to 0.028 (P<0.05). Whilst soluble 4-1BB levels weresimilar in control and group A patients, significantly elevated levelswere found in the plasma of group B patients (see FIG. 4).

[0285] These data identify a change in the circulating cytokine balancetowards a pro-inflammatory (Th1) environment in the plasma of donorsassociated with early recipient death post-transplantation. Elevationsin the levels of IL-17 and IL-18 are predominantly responsible for this.IL-18 utilizes the same signaling pathway as IL-1 and lipopolysacharide(Sims, J E, Curr Opin Immunol. 2002;14(1):117-22), both of which arenegatively inotropic. IL-17 induces nitric oxide production from avariety of cells, and also stimulates production of a number ofcytokines and prostaglandins from a variety of cell types. The elevatedlevels of IL-17 and IL-18 seen, in particular, in group B patients couldwell contribute to the adverse recipient outcome early aftertransplantation. Significantly elevated levels of soluble 4-1BB werealso observed in the plasma samples of donors in group B, suggestingthat enhanced antigen presenting cell:T-cell interaction may occur inthese patients.

[0286] Analysis of donor cytokine expression and levels of moleculessuch as IL-17, IL-18 and 4-1BB is valuable in identifying hearts fromdonors that may require intensive supportive therapypost-transplantation, or indeed that should not be used fortransplantation. These data emphasize the importance of immuneactivation in brain death, its potential impact on outcomepost-transplantation and the idea that therapies aimed at altering thedonor cytokine balance may result in improved recipient outcome. Theseresults suggest that elevated 4-1BB, as well as other cytokines such asIL-17 and IL-18, primes the heart for rejection in a recipient soonafter transplantation or that these 4-1BB, IL-17 and/or IL-18 maydirectly mediate impaired myocardial performance. Thus, the studiespresented herein demonstrate that IL-17, IL-18 and 4-1BB are implicatedin cardiovascular disease and provides a basis for treatingcardiovascular disease by administering IL-17, IL-18 and/or 4-1BBantagonists, alone or in combination. TABLE 2 IL-4 IL-5 IL-10 IL-13 IFNγIL-12 IL-15 IL-17 IL-18 4-1BB Control  2.6 0.9  1.7 1.9  1.9 10.4 38.7 1.1  68.1  30 mean (SE) (0.9) (0.3) (0.3) (0.7) (0.4) (4.7) (13.85)(0.5) (23) (20) Group A 14 2.4 19.9 8.8 20.7 41.5  8.6  18 186  90 mean(SE) (5.3) (1.6) (4.8) (5.9) (8.6) (31.6) (3.1) (18) (50) (80) Group B 3.5 0.45  7 1.1  8.4  7.4  5.4 292 373 420 mean (SE) (0.4) (0.1) (2)(0.2) (1.5) (1.9) (1.8) (70) (51) (170)

Example 3 IL-17 Levels and Heart Chamber Dimensions

[0287] These studies demonstrate that exposure to IL-17 results in adrop in left ventricle dimensions.

[0288] Female C57/Black6 mice were anethetised with avertin. A midlinelaparotomy was performed and the inferior vena cava was carinulated. Abase line echocardiogram was then performed. Human IL-17 (200 ng) wasthen administered in a bolus of 200 ml of PBS (pH 7.4). At the indicatedtime points the echocardiogram was repeated and left ventricularinternal diameter in diastole and systole were determined.

[0289] As shown in FIG. 4, these data demonstrate that IL-17, followingacute administration, results in diminished chamber dimensions withmaintained ejection fraction. The reduction in diastolic dimensions maysuggest that IL-17 plays some part in mediating diastolic dysfunction,though it may also be involved in systolic heart failure. This datasuggests IL-17 is implicated in cardiovascular disease and provides abasis for treating cardiovascular disease by administering IL-17antagonists.

Example 4 IL-17 and IL-18 Levels are Elevated in a Murine Myosin-InducedMyocarditis Model

[0290] This study shows that IL-17 and IL-18 are elevated in anexperimental autoimmune myocarditis (EAM) model, which shows a similarcourse of disease as seen in humans. The terms “EAM” and “cardiacmyosin-induced myocarditis” are used interchangeably to describe similarmodels.

[0291] It is well known in the art that myocarditis is associated withan autoimmune process in which cardiac myosin is a major autoantigen.Cardiac myosin-induced myocarditis histologically resemblesviral-induced myocarditis. It is generally agreed that both antibody andT-cells are implicated in inflammatory heart disease, such asmyocarditis. Experimental autoimmune myocarditis models that mimic thedisease in humans have been developed in a variety of rodent models andare well known in the art (see, for example, in A/J mice: Neu, N, etal., J. Immunol. 1987, 139:3630-3636 and Smith, S C, et al., J. Immunol.1991, 147:2141-2147; in BALB/c mice: Pummarer, C L, et al., J. Clin.Invest. 1996, 97:2057-2062 and Liao, L, et al., J. Clin. Invest. 1993,92:2877-2882; and in Lewis rats: Kodama, M, et al., Clin. ImmunolImmunopath. 1991, 57:250-262 and Wegmann, K W, et al., J. Immunol. 1994,153:892-900).

[0292] It has been shown in the A/J mouse EAM model that blocking IL-4with anti-IL-4 monoclonal antibody reduced the severity of EAM byshifting the immune response from a Th2-like response to a Th1-likeresponse with a concomitant increase in INF-γ production, whichsuggested INF-γ limits the disease. Blockade of INF-γ was shown toexacerbate disease, thereby establishing the basis for using an INF-γknockout (INF-γ^(−/−)) in a EAM model (Afanasyeva, M, et al., Am JPathol 2001, 159:193-203).

[0293] Autoimmune myocarditis was induced in mice by immunizing BALB/cand INF-γ^(−/−) knockout mice with 200 ug α-myosin heavy chain plus MTB(mycobacterium tuberculosis was included at 5 mg/ml H37Ra; Difco/BectinDickinson, Franklin Lakes, N.J.) and Pertussis toxin at 500 ng (Listbiological laboratories, Campbell, Calif.) intraperitoneally in a volumeof 400 ul. Interferon gamma-deficient mice were obtained from JacksonLaboratories, Bar Harbor, ME. BALB/c and INF-γ^(−/−) were boosted on Day7 by immunization with myosin formulated in Complete Freund's Adjuvant(CFA) with omission of Pertussis toxin. Animals were sacrificed at days35, 55 or 85 and the following analysis were preformed: histology,anti-myosin antibody titre, serum cytokine profile and antigen specificT-cell proliferation assays. Some animals underwent echocardiography.

[0294] BALB/c and INF-γ^(−/−) mice that were immunized with α-myosinheavy chain developed antibodies against myosin peptide as measured bystandard ELISA techniques. Myosin-immunized BALB/c and INF-γ^(−/−) micedeveloped myocardial lesions, with the knockout mice showing an increasein lesion number and severity over negative controls and BALB/c mice. Inaddition, INF-γ knockout mice showed a greater degree of myocardialinflammation and fibrosis, as well as a greater percent increase inheart/body weight ratio over negative controls and BALB/c mice.Mysoin-specific T-cell proliferation responses were shown inmyosin-immunized INF-γ knockout and BALB/c mice, as measure by standardthymidine incorporation assays.

[0295] Furthermore, plasma levels of IL-17 and IL-18 were elevated inthe cardiac myosin-induced myocarditis model. IL-17 and IL-18 wereassayed using commercially available ELISA kits, such as described inExample 1. As shown in FIG. 5A, IL-17 levels were markedly increased inINF-γ^(−/−) mice at day 28 and 35 post immunization. Plasma levels ofIl-18 rose sharply at 9 days post immunization and remained elevated outto day 28 (FIG. 5B). This data clearly shows that circulating plasmalevels of IL-17 and IL-18 are elevated in the myosin-induced model andthat IL-17 and IL-18 are associated with myocarditis immunopathology.This data provides a basis for treating cardiovascular disease byadministering IL-17 and/or IL-18 antagonists, alone or in combination.

Example 5 IL-17 Expression in T-Cells in the Myosin-Induced MyocarditisModel

[0296] These experiments demonstrate that IL-17 is expressed at highlevels in T-cell populations isolated from EAM mice. Details of the EAMmodel are provided in the previous Example.

[0297] T-cells were isolated from EAM mice using standard techniques andstimulated with anti-CD3 antibody. As shown in FIG. 6A, the animalimmunized with cardiac myosin having histologically demonstratedcardiopathology (animal B) had significantly higher expression levels ofIL-17 over negative controls (animals C and D), as well as an animalimmunized with cardiac myosin, but not exhibiting signs ofcardiopathology (animal A).

[0298] IL-17 expression in EAM mice was shown to be a myosin-specificT-cell response. On average, IL-17 levels were approximately 25 foldhigher in T-cells isolated from animals immunized with cardiac muyosinand exposed to antigen presenting cells fed myosin over antigenpresenting cells not exposed to myosin (FIG. 6B).

[0299] In a related study, the antigen presenting cells were exposed toα-myosin peptide rather than being fed whole α-myosin protein. As in theprevious study, T-cells from the animal immunized with cardiac myosinand having histological evidence of cardiopathology (animal B) releasedsurprisingly high levels of IL-17 in response to antigen-specificstimulation by peptide-pulsed antigen presenting cells (FIG. 6C).Without being bound by theory, this study suggests that activated(likely CD4+) T-cells when encountering antigen presenting cells bearingheart antigens proliferate and release IL-17. The released IL-17 maycontribute to inflammatory cell infiltration into the heart, directmyocardial damage or may also have a direct depressant effect on heartfunction.

Example 6 IL-18 and IL-18 Binding Protein Plasma Levels in AcuteCoronary Syndrome Patients

[0300] These studies show that IL-18 is elevated in patients havingacute coronary syndrome and that IL-18 correlates with increased riskfor major adverse cardiac events (MACEs).

[0301] Patients were stratified into three patient groups: those havingstable coronary artery disease (CAD), acute coronary syndrome (ACS) withcardiac troponin I (cTnI) plasma levels less than 0.4 ng/ml or acutecoronary syndrome (ACS) with cardiac troponin I (cTnI) plasma levelsgreater than 0.4 ng/ml. The CAD group included patients having stableangina and the ACS group included patients having unstable angina,non-ST elevation myocardial infarction, ST elevation myocardialinfarction and sudden ischemic death.

[0302] Cardiac troponin I is recognized as a reliable biochemical markerfor the diagnosis of myocardial injury, such as myocardial necrosisresulting from ischemia. Elevated cardiac troponin I is stronglyassociated with a high-risk profile for short and long term adversecardiac events. Measuring the relative levels of cardiac troponin Iprovides a reliable stratification of risk and prediction of outcome foracute coronary syndrome patients. In this study, patients having acardiac troponin I level of greater than 0.4 ng/ml suffered a seriousadverse cardiac event, often resulting in death, within ten days.

[0303] Cytokine levels were measured in plasma samples from patientsfrom each of the three groups essentially as described in the protocolsprovided in commercially available ELISA kits (see, for example,QUANTKINE® R&D Systems, Minneapolis, Minn., which provides assays forthe quantitative determination of human IL-18). IL-18 Binding Protein A(IL-18 Bpa) was measured using commercially available antibodies fromR&D. Plates were coated with a capture antibody. Samples were then addedto wells and incubated at room temperature for 2 hours. Wells were thenwashed, incubated with a biotinylated detection antibody and theimmunoreaction detected using standard techniques and TMB as chromogen.IL-18 Binding Protein produced in house was used as standard.

[0304] As shown in FIG. 8A, plasma levels of IL-18 was elevated in theACS group having a cTnI level of less than 0.4 ng/ml, and moresignificantly, IL-18 levels were even higher in patients in the ACSgroup having a cTnI level of greater than 0.4 ng/ml. FIG. 8B shows theratio of IL-18:IL-18 BPa among the three groups and illustrates thatIL-18 Binding Protein levels are not elevated in either the ACS groups,which demonstrates that the elevated IL-18 levels are unopposed by thisimmune mechanism.

[0305] This data shows that circulating levels of IL-18 are elevated inACS patients at high risk of subsequent major adverse cardiac events andthat IL-18 Binding Protein levels do not rise concomitantly tocounteract the immune response. Thus, IL-18 is implicated incardiovascular disease and provides a basis for treating cardiovasculardisease by administering IL-18 antagonists, alone or in combination withother antagonists described herein.

[0306] In addition, elevated IL-18 levels correlate with elevated cTnIlevels and disease progression or disease severity. Therefore, IL-18 mayalso serve as a surrogate marker for increased risk for serious adversecardiac events. Embodiments of the present invention include diagnosticassays for determining the level of IL-18 in patients havingcardiovascular disease for the purpose of assessing disease progressionor severity.

Example 7 4-1BB Ligand Knockout Mice are Protected in andAdriamycin®-Induced Murine Model of Dilated Cardiomyopathy

[0307] These experiments demonstrate that 4-1BB-L knockout mice(4-1BB-L^(−/−) or 4-1BB-L KO) showed no mortality and had a delayedonset of cardiac dysfunction in an Adriamycin®-induced murine model ofdilated cardiomyuopathy.

[0308] Adriamycin® (Doxorubicin Hydrochloride, an anthracyclineantibiotic-Pharmacia, Milan, Italy) has been shown to exhibit myocardialtoxicity resulting in congestive heart failure, i.e., ischemic ordilated cardiomyopathy. Many animal models have been developed over theyears using using Adriamycin® and are well-known in the art.

[0309] Male 4-1BB-L^(−/−) (53 days old) and C57B1/6 (59 days old,Taconic, Germantown, N.Y.) were used in the study. Mice wereanesthetized with 100-150 ul ketamine-xylazine, weighed and ear tagged.Baseline echocardiographic (echo) measurements were taken from theparasternal long axis view while the mouse lay prone on a 1 cm thickagarose gel pad (SonosS500—Philips Co., with a S12 probe and a Instecheated microscope stage with 1 cm thick 1% agarose gelpad). Measurementstaken included: AoR diameter, LA dimension, ACS, RVd, IVSd, LVIDd,LVPWd, IVSs, LVIDs, LVPWs, and HR. Echo evaluations were perfomred atweeks 0, 2.5, 5 and 7 and a phenotype assigned to each mouse based onthe weekly echo measurements. Fractional shortening (FS) was calculatedby the echo instrument. Measurements were normalized between mice byobtaining a PSLA image where the AoR and LA dimensions had a 1:1 ratio.Additionally, LV dimensions were taken from a Mmode image obtained bydissecting the LV perpendicularly through the PW, just distal to thetips of the mitral valve leaflets (sweep speed 100). Diastolicmeasurements were taken at the peak of the QRS complex of the EKG, whilesystolic measurements were recorded at the point of maximal contractionof the PW. All measurements were recorded onto optical disk, videotapedand printed.

[0310] After all echo measurements were obtained, the mice were injectedretroorbitally with 22.5 mg/kg Adriamycin® (Sigma-Aldrich/Fluka, St.Louis, Mo.) and allowed to recover from anesthesia. Adriamycin wasprepared from a powdered stock by dissolving 10 mg in 1 ml sterile waterfor a stock concentration of 10 mg/ml. Further dilutions were made withsterile saline. Solutions were made on the day of injection. The micethat received a dose of 22.5 mg/kg were injected with 150 ul of a 3.75mg/ml working dilution for a total dose of 0.5625 mg. Body weights wererecorded daily.

[0311] If an animal was sacrificed due to a greater than 15% loss inbody weight or at the end of the study, the hearts were harvested andfixed in 10% NBF (neutral buffered formalin). Briefly, thoracicinsicions were made to expose the heart. Using a 27 g needle, 1M KCl(final concentration=50 mM) was injected directly into the heart to stopthe hearts in diastole; PBS was then injected directly into the heart toflush blood out of the chambers. The heart was carefully removed, rinsedin PBS and placed into 10% NBF.

[0312] As mentioned above, a phenotype was assigned to each mouse basedon the echo results and each mouse was classified according to thedegree of cardiotoxicity into stages I, II, III or IV. The followingcriteria was used to phenotype the mice: Diastolic Dysfunction: LVIDd %Δ and LVIDs % Δ reduced >10% and FS same or increased; SystolicDysfunction: LVIDd % Δ plus LVIDs % Δ increased >25% and FSreduced >12%; Diastolic and Systolic Dysfunction: LVIDd % Δ plus LVIDs %Δ reduced >20% and FS reduced >12%; Dilated: LVIDd % Δ >12% and LVIDs %Δ >12%.

[0313] 4 of 10 wild type mice died between 8 and 10 days afterAdriamycin® injection. These deaths are considered chronic heart failuredeaths. Wild-type mice challenged with Adriamycin® had an approximate2-week mortality rate of 50% and approximately 66% of the mice exhibitedevidence of cardiac dysfunction 2 to 2.5 weeks postAdriamycin®-challenge. The wild-type survivors generally showed twophenotypes: (a) systolic dysfunction with LV chamber dilation withdiminished ejection fraction (EF), and (b) diastolic disfunction withprogressive reduction in chamber dimensions with diminished ventricularfilling but maintained EF.

[0314] In contrast, no mortality was observed in the 4-1BB-L KO mice andnone of the 4-1BB-L KO mice showed signs of the most severecardiotoxicity following Adriamycin® challenge compared to 59 to 71% ofwild type mice. TABLE 3 4-1BB-L KO Wild Type B16 Historical B16 cntls NoDysfunction 7/14 (50%) 2/7 (29%)  9/22 (41%) (Class I) Transient 1/14(7%) 0/7 (0%)  1/22 (5%) Dysfunction (Class II) Progressive 6/14 (43%)1/7 (14%)  5/22 (23%) Dysfunction (Class III) Chronic Failure 0/14 (0%)4/7 (57%)  7/22 (32%) (Class IV) Total cardiotoxicity 7/14 (50%) 5/7(71%) 13/22 (59%)

[0315] These results demonstate that the 4-1BB and 4-1BB-Lreceptor:ligand pair are implicated in immune responses associated withcardiovascular disease, and in particular play a role in ischemiccardiomyopathy. These data establish a sound basis for preventing,treating or alleviating the symptoms of cardiovascular disease, and inparticular ischemic cardiomyopathy, by antagonizing the interaction of4-1BB and 4-1BB-L. Furthermore, these results demonstrate thatantagonizing 4-1BB-L:4-1BB interactions reduces Adriamycin®-inducedcardiotoxicity. Therefore, 4-1BB, CD30 and/or OX40 antagonists, may beused prior to or in combination with chemotherapeutic compositions inorder to reduce the cardiotoxic side-effects of such chemotherapeutics.

Example 8 4-1BB is Expressed by Damaged Cardiac Interstitial Cells and4-1BB/4-1BBL Signaling Contributes to Adriamycin®-Induced Cardiomyopathyin Mice

[0316] Co-stimulatory pathways have been implicated in myocarditis anddilated cardiomyopathy in mice and humans. Expression of co-stimulatoryligands is increased on cardiac myocytes, while the receptors areexpressed on infiltrating immune/inflammatory cells (Seko et al, 2002;Seko et al, 2001; Seko et al, 1998). The aim of these studies was todetermine the role of the 4-1BB/4-1BBL co-stimulatory pathway in theonset and progression of Adriamycin®-induced cardiomyopathy, which isnot associated with the extensive inflammatory infiltrate seen withother forms of dilated cardiomyopathy. These studies demonstrate a rolefor the 4-1BB/4-1BBL immune co-stimulatory pathway inAdriamycin®-induced cardiomyopathy, deomonstrated a novel cardiacexpression pattern of 4-1BB and implicated apoptosis as a mechanism ofco-stimulatory contribution to Adriamycin®-induced cardiomyopathy.

[0317] 1. 4-1BB and 4-1BBL Expression is Up-Regulated inAdriamycin®-Treated Myocardium.

[0318] Six-week old C57B1/6 mice were injected retrorbitally with 45mg/kg Adriamycin® and the hearts were collected at 0, 24, 48, 72, and 96hrs post treatment. Immunohistochemical staining on cryo-sections forCD45 was performed to detect inflammatory infiltrate, as well asstaining for 4-1BB. Immunohistochemistry of CD45 did not recognizepositive cells in Adriamycin®-treated myocardium. As positive control,CD45 positive cells were detected in spleen. While no inflammatoryinfiltrate was observed in the Adriamycin®-cardiomyopathy, we foundexpression of 4-1BB induced on 1-5% of cardiac interstitial cells within2 days after Adriamycin® administration. As a percentage of animalsdemonstrating 4-1BB positive cells, 50% mice were positive for 4-1BB inmyocardium at 48 hrs and 75% at 72 hrs. In immunohistochemical analysis,4-1BBL increased in positivity after Adriamycin® treatment. Leukocytecounts in wild type and 4-1BBL−/− demonstrated similar level ofneutrophils, monocytes and lymphocytes in peripheral blood, indicatingthe improvement of cardiac function by loss of 4-1BBL is unrelated tohematopoietic and inflammatory changes.

[0319] 2. Treatment with Agonistic Anti-4-1BB Antibody (M6) AcceleratedCardiac Dysfunction and m4-1BB-Fc Delayed and Reduced CardiacDysfunction.

[0320] An agonistic antibody to 4-1BB (M6) accelerated and exacerbatedcardiac dysfunction in an Adriamycin® challenge model. In contrast,m4-1BB-Fc, which is a soluble decoy receptor for 4-1BBL, delayed andreduced cardiac dysfunction.

[0321] 100 ug/mouse of agonistic antibody to 4-1BB (M6, lot 9159-069from Amgen Hybrodima Group) was administered IP on days 0, 3 and 6. Day0 being the date of Adriamycin® challenge. NaCl controls were includedfor each treatment regimen. Wild type (WT), 4-1BBL KO, or WT micetreated with an activating antibody to 4-1BB (M6) were challenged withAdriamycin® in doses ranging from 22.5 to 45 mg/kg by retroorbitalinjection, immediately following baseline echocardiographic (ECHO)analysis of cardiac function. Serial ECHO analysis was performed at 1,2.5 and/or 5 weeks after Adriamycin® challenge. In one study, WT and4-1BBL KO mice were challenged with Adriamycin® (45 mg/kg) and heartswere collected at 24, 48 and 72 hours. TUNEL positivity by IHC andcaspase 3 activation by western blot were determined as indices ofapoptosis. 4-1BBL KO mice had improved cardiovascular function anddecreased penetrance of cardiomyopathy, as evaluated by M-modeechocardiography compared to WT controls (see Table 4, below and FIG.10). For Table 4, combination of stroke volume, systolic, diastolic endvolume was used to categorize functional phenotype. 4-1BBL KO mice havereduced cardiotoxicity and improved function post-Adriamycin®. Treatmentof WT C57B1/6 mice with the 4-1BB activating antibody (M6) acceleratedthe onset of ADR-induced cardiomyopathy (FIG. 11). TABLE 4 Phenotype WT4-1BB-/- KO M6 antibody M4-1BB-Fc Normal 48% 79% 53% 70% Dysfunction 52%21% 47% 30%

[0322] 3. Apotposis was Increased in Adriamycin Myocardium.

[0323] C57B1/6 mice were treated with 45 mg/kg adriamycin. Heart tissueswere harvested at different time points and analyzed for TUNEL positivenuclei. TUNEL positivity increased at 48 hrs and peaked at 72 hrs. Wholeheart digests were collected from Adriamycin®-treated mice were stainedwith troponin-I(TnI) antibody and FITC conjugated secondary antibody andpropidium iodide. TnI positive cells were analyzed for sub-G1 DNAfragments. In WT mice, cardiac TUNEL positivity increased at 48 and 72hr after Adriamycin® injection.

[0324] Cardiac apoptosis, measured by TUNEL and sub-G1 DNA, wasincreased 3 days after Adriamycin®, concomitant with the increasedexpression of 4-1BB on interstitial cells. Chronic ongoing apoptosis,determined 5 weeks after Adriamycin® challenge when cardiac dysfunctionis maximal in wild type but largely absent in 4-1BBL−/− mice, was lowerin 4-1BBL−/− mice (1.5-fold vs baseline), compared to WT mice (4 fold).In a separate study, caspase 3 activation, determined by Western blot,was increased at 48 to 72 hrs post-Adriamycin® (45 mg/kg). In contrast,Adriamycin® did not induce caspase 3 cleavage in 4-1BBL−/− myocardium.Determined by western blot, Adriamycin® reduced phosphorylation of Aktin wild type but not 4-1BBL−/− hearts. Phosphorylation of JNK and p38was not impacted by Adriamycin®. Therefore, 4-1BB/4-1BBL immuneco-stimulatory pathway contributes to Adriamycin®-inducedcardiomyopathy, possibly through modulation of Akt signaling to regulate

[0325] In conclusion, 4-1BBL deficient mice and 4-1BBL decoyreceptor-treated mice conferred partial resistance to adriamycin inducedcardiac damage, whereas 4-1BB activating antibody accelerated onset ofdamage, implying the contribution of 4-1BB to Adriamycin® effects inheart. Apoptosis, measured by TUNEL, sub-G1 DNA and activated caspase-3,was increased in Adriamycin®-treated wild type myocardium, but reducedin 4-1BBL−/−. Phosphorylation of Akt was selectively suppressed byAdriamycin®, but maintained by loss of 4-1BBL, indicating the modulationof apoptosis by co-stimulatory pathway in heart is possibly through Akt,but not Jnk and p38 signaling. The consistency of decreased index ofapoptosis and the improved cardiac function in 4-1BBL−/− suggestsapoptosis play a pivotal role in Adriamycin®-induced cardiac deficiency.

Example 9 Antibodies Generated Against IL-17, IL-17R, IL-18, IL-18R,4-1BB, 4-1BB-L, CD30, CD30-L and CD39

[0326] Monclonal and/or polyclonal antibodies were generated againstIL-17, IL-17R, IL-18, IL-18R, 4-1BB, 4-1BB-L, OX40, CD30, CD30-L andCD39 using standard techniques. One or more of these antibodies, orother antibodies directed against IL-17, IL-17R, IL-18, IL-18R, 4-1BB,4-1BB-L, OX40, OX40-L, CD30, CD30-L or CD39 may be used as an antagonistfor the treatment of cardiovascular disease. Immunogens used to generateantibodies included purified polypeptides, fragment thereof such as theextracellular domain, Fc-fusion proteins of the extracellular domains,and leucine-zipper derivatives of the extracellular domains (refer toTable 3 below). Of course, other forms of the proteins could be used togenerate antibodies, such as any immunogenic fragment, alone or fusedwith other proteins. In addition, DNA encoding a polypeptide can be usedas an immunogen, for example, DNA may be given intradermally (Raz etal., 1994, Proc. Natl. Acad. Sci. USA 91: 9519) or intamuscularly (Wanget al., 1993, Proc. Natl. Acad. Sci. USA 90: 4156); saline has beenfound to be a suitable diluent for DNA-based antigens, or by othersimilar techniqes, as reviewed by Pardoll and Beckerleg in Immunity 3:165, 1995.

[0327] In general, the antibodies were generated by the followingmethod: rodents (BALB/c mice or Lewis rats, for example) were immunizedwith the polypeptide immunogen emulsified in an adjuvant (such ascomplete or incomplete Freund's adjuvant, alum, or another adjuvant,such as Ribi adjuvant R700 (Ribi, Hamilton, M T)), and injected inamounts ranging from 10-100 micrograms subcutaneously orintraperitoneally. Ten days to three weeks days later, the immunizedanimals were boosted with additional immunogen and periodically boostedthereafter on a weekly, biweekly or every third week immunizationschedule.

[0328] Serum samples were periodically taken by retro-orbital bleedingor tail-tip excision to test for polypeptide-specific antibodies bydot-blot assay, ELISA (enzyme-linked immunosorbent assay),immunoprecipitation, or other suitable assays, such as FACS analysis ofantibody binding to its original immunogen. Following detection of anappropriate antibody titer, positive animals were provided one lastintravenous injection of respective immunogen in saline. Three to fourdays later, the animals were sacrificed, spleen cells harvested andfused to a murine myeloma cell line, e.g., NS1 or preferablyP3X63Ag8.653 (ATCC CRL-1580). The hybridoma cells were plated inmultiple microtiter plates in a HAT (hypoxanthine, aminopterin andthymidine) selective medium to inhibit proliferation of non-fused cells,myeloma hybrids, and spleen cell hybrids.

[0329] Positive hybridoma cells were injected intraperitoneally intosyngeneic rodents to produce ascites containing high concentrations ofmonoclonal antibodies. Alternatively, hybridoma cells were be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies were purified by ammonium sulfate precipitation, followed bygel exclusion chromatography. Alternatively, affinity chromatographybased upon binding of antibody to protein A or protein G has also beenused.

[0330] Of course other conventional techniques may be used, such asthose described in U.S. Pat. No. 4,411,993. For example, the immunogenpreparation, choice of adjuvant and immunization protocol are well knownin the art and may be found, for example in Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988). TABLE 4 Molecule Ab name ImmunogenSpecies Type/Isotype IL-17 mIL-17-M210 mCTLA8-Fc Rat mAb IL-17 mIL-17-P1mIL-17 Rabbit polyclonal IL-17R hIL-17R-M202 hIL-17R-Fc (cos) MouseIgG2a IL-17R hIL-17R-M203 hIL-17R-Fc (cos) Mouse IgG1 IL-17RhIL-17R-M204 hIL-17R-Fc (cos) Mouse IgG2a IL-17R mIL-17R-M177 mIL-17R-FcRat IgG2a IL-17R mIL-17R-M178 mIL-17R-Fc Rat IgG2a IL-17RH mIL-17RH-M561mIL-17RH-Fc Rat IgG2a IL-17RH mIL-17RH-M561 mIL-17RH-Fc Rat IgG2a IL-18hIL-1R/AcpL-P1 hIL-1R/AcpL-Fc Rabbit Polyclonal IL-18 mIL-1R/AcpL-P1mIL-1R/AcpL-Fc Rabbit Polyclonal IL-18R hIL-18R-M495 hIL-18R Rat IgG2bIL-18R hIL-18R-M496 hIL-18R Rat un- determined IL-18R hIL-18R-M497hIL-18R Rat IgG2b IL-18R mIL-18R-M375 mRp1-Fc Rat IgG1 IL-18RmIL-18R-M376 mIL-1R/RpL-Fc Rat IgG2a IL-18R mIL-18R-M377 mIL-1R/RpL-FcRat IgG1 IL-18R hIl-18R-P1 h2F1 GST Rabbit polyclonal IL-18R hIl-18R-P1sol m2F1 Rabbit polyclonal 4-1BB h41BB-M121 h4-1BB-Fc Mouse mAb/IgG14-1BB h41BB-M127 h4-1BB-Fc Mouse mAb/IgG1 4-1BB h41BB-M135 h4-1BB-FcMouse mAb/IgG1 4-1BB h41BB-M4 sol h4-1BB-Fc Mouse mAb/IgM 4-1BB h41BB-M8sol h4-1BB-Fc Mouse mAb/IgG3 4-1BB h41BB-M6 sol m4-1BB-Fc Rat mAb/IgG2a4-1BB m41BB-P1 sol m4-1BB-Fc Rabbit polyclonal 4-1BB-L m41BBL-M520m4-1BBL-leuzip Rat mAb/IgG2a 4-1BB-L m41BBL-P2 flag m4-1BBL Rabbitpolyclonal CD30 hCD30-M44 hCD30-Fc Mouse mAb/IgG1 CD30 hCD30-M67hCD30-Fc Mouse mAb/IgG1 CD30-L hCD30L-M80 hCD30L-Fc/CV-1 Mouse mAb/IgG2bCD30-L hCD30L-M81 hCD30L-Fc/CV-1 Mouse mAb/IgG2b CD30-L hCD30L-M82hCD30L(CHO) Mouse mAb/IgG2a CD30-L mCD30L-M15 mCD30L(CHO) Rat mAb/IgG2aCD30-L mCD30L-M30 mCD30L(CHO) Rat mAb/IgG2a OX40 mOX40-M5 mOX40-Fc RatIgG1 OX40 mOX40-M6 mOX40-Fc Rat IgG2b CD39 mCD39-M105 Rat mAb/IgG2a

[0331]

1 36 1 1874 DNA Homo sapiens CDS (54)..(518) 1 gaattccggc aggcacaaactcatccatcc ccagttgatt ggaagaaaca acg atg 56 Met 1 act cct ggg aag acctca ttg gtg tca ctg cta ctg ctg ctg agc ctg 104 Thr Pro Gly Lys Thr SerLeu Val Ser Leu Leu Leu Leu Leu Ser Leu 5 10 15 gag gcc ata gtg aag gcagga atc aca atc cca cga aat cca gga tgc 152 Glu Ala Ile Val Lys Ala GlyIle Thr Ile Pro Arg Asn Pro Gly Cys 20 25 30 cca aat tct gag gac aag aacttc ccc cgg act gtg atg gtc aac ctg 200 Pro Asn Ser Glu Asp Lys Asn PhePro Arg Thr Val Met Val Asn Leu 35 40 45 aac atc cat aac cgg aat acc aatacc aat ccc aaa agg tcc tca gat 248 Asn Ile His Asn Arg Asn Thr Asn ThrAsn Pro Lys Arg Ser Ser Asp 50 55 60 65 tac tac aac cga tcc acc tca ccttgg aat ctc cac cgc aat gag gac 296 Tyr Tyr Asn Arg Ser Thr Ser Pro TrpAsn Leu His Arg Asn Glu Asp 70 75 80 cct gag aga tat ccc tct gtg atc tgggag gca aag tgc cgc cac ttg 344 Pro Glu Arg Tyr Pro Ser Val Ile Trp GluAla Lys Cys Arg His Leu 85 90 95 ggc tgc atc aac gct gat ggg aac gtg gactac cac atg aac tct gtc 392 Gly Cys Ile Asn Ala Asp Gly Asn Val Asp TyrHis Met Asn Ser Val 100 105 110 ccc atc cag caa gag atc ctg gtc ctg cgcagg gag cct cca cac tgc 440 Pro Ile Gln Gln Glu Ile Leu Val Leu Arg ArgGlu Pro Pro His Cys 115 120 125 ccc aac tcc ttc cgg ctg gag aag ata ctggtg tcc gtg ggc tgc acc 488 Pro Asn Ser Phe Arg Leu Glu Lys Ile Leu ValSer Val Gly Cys Thr 130 135 140 145 tgt gtc acc ccg att gtc cac cat gtggcc taagagctct ggggagccca 538 Cys Val Thr Pro Ile Val His His Val Ala150 155 cactccccaa agcagttaga ctatggagag ccgacccagc ccctcaggaaccctcatcct 598 tcaaagacag cctcatttcg gactaaactc attagagttc ttaaggcagtttgtccaatt 658 aaagcttcag aggtaacact tggccaagat atgagatctg aattacctttccctctttcc 718 aagaaggaag gtttgactga gtaccaattt gcttcttgtt tacttttttaagggctttaa 778 gttatttatg tatttaatat gccctgagat aactttgggg tataagattccattttaatg 838 aattacctac tttattttgt ttgtcttttt aaagaagata agattctgggcttgggaatt 898 ttattattta aaaggtaaaa cctgtattta tttgagctat ttaaggatctatttatgttt 958 aagtatttag aaaaaggtga aaaagcacta ttatcagttc tgcctaggtaaatgtaagat 1018 agaattaaat ggcagtgcaa aatttctgag tctttacaac atacggatatagtatttcct 1078 cctctttgtt tttaaaagtt ataacatggc tgaaaagaaa gattaaacctactttcatat 1138 gtattaattt aaattttgca atttgttgag gttttacaag agatacagcaagtctaactc 1198 tctgttccat taaaccctta taataaaatc cttctgtaat aataaagtttcaaaagaaaa 1258 tgtttatttg ttctcattaa atgtatttta gcaaactcag ctcttccctattgggaagag 1318 ttatgcaaat tctcctataa gcaaaacaaa gcatgtcttt gagtaacaatgacctggaaa 1378 tacccaaaat tccaagttct cgatttcaca tgccttcaag actgaacaccgactaaggtt 1438 ttcatactat tagccaatgc tgtagacaga agcattttga taggaatagagcaaataaga 1498 taatggccct gaggaatggc atgtcattat taaagatcat atggggaaaatgaaaccctc 1558 cccaaaatac aagaagttct gggaggagac attgtcttca gactacaatgtccagtttct 1618 cccctagact caggcttcct ttggagatta aggcccctca gagatcaacagaccaacatt 1678 tttctcttcc tcaagcaaca ctcctagggc ctggcttctg tctgatcaaggcaccacaca 1738 acccagaaag gagctgatgg ggcagaatga actttaagta tgagaaaagttcagcccaag 1798 taaaataaaa actcaatcac attcaattcc agagtagttt caagtttcacatcgtaacca 1858 ttttcgcccg gaattc 1874 2 155 PRT Homo sapiens 2 Met ThrPro Gly Lys Thr Ser Leu Val Ser Leu Leu Leu Leu Leu Ser 1 5 10 15 LeuGlu Ala Ile Val Lys Ala Gly Ile Thr Ile Pro Arg Asn Pro Gly 20 25 30 CysPro Asn Ser Glu Asp Lys Asn Phe Pro Arg Thr Val Met Val Asn 35 40 45 LeuAsn Ile His Asn Arg Asn Thr Asn Thr Asn Pro Lys Arg Ser Ser 50 55 60 AspTyr Tyr Asn Arg Ser Thr Ser Pro Trp Asn Leu His Arg Asn Glu 65 70 75 80Asp Pro Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys Cys Arg His 85 90 95Leu Gly Cys Ile Asn Ala Asp Gly Asn Val Asp Tyr His Met Asn Ser 100 105110 Val Pro Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu Pro Pro His 115120 125 Cys Pro Asn Ser Phe Arg Leu Glu Lys Ile Leu Val Ser Val Gly Cys130 135 140 Thr Cys Val Thr Pro Ile Val His His Val Ala 145 150 155 33223 DNA Homo sapiens CDS (93)..(2693) 3 gggagaccgg aattccgggaaaagaaagcc tcagaacgtt cgctcgctgc gtccccagcc 60 ggggccgagc cctccgcgacgccacccggg cc atg ggg gcc gca cgc agc ccg 113 Met Gly Ala Ala Arg SerPro 1 5 ccg tcc gct gtc ccg ggg ccc ctg ctg ggg ctg ctc ctg ctg ctc ctg161 Pro Ser Ala Val Pro Gly Pro Leu Leu Gly Leu Leu Leu Leu Leu Leu 1015 20 ggc gtg ctg gcc ccg ggt ggc gcc tcc ctg cga ctc ctg gac cac cgg209 Gly Val Leu Ala Pro Gly Gly Ala Ser Leu Arg Leu Leu Asp His Arg 2530 35 gcg ctg gtc tgc tcc cag ccg ggg cta aac tgc acg gtc aag aat agt257 Ala Leu Val Cys Ser Gln Pro Gly Leu Asn Cys Thr Val Lys Asn Ser 4045 50 55 acc tgc ctg gat gac agc tgg att cac cct cga aac ctg acc ccc tcc305 Thr Cys Leu Asp Asp Ser Trp Ile His Pro Arg Asn Leu Thr Pro Ser 6065 70 tcc cca aag gac ctg cag atc cag ctg cac ttt gcc cac acc caa caa353 Ser Pro Lys Asp Leu Gln Ile Gln Leu His Phe Ala His Thr Gln Gln 7580 85 gga gac ctg ttc ccc gtg gct cac atc gaa tgg aca ctg cag aca gac401 Gly Asp Leu Phe Pro Val Ala His Ile Glu Trp Thr Leu Gln Thr Asp 9095 100 gcc agc atc ctg tac ctc gag ggt gca gag tta tct gtc ctg cag ctg449 Ala Ser Ile Leu Tyr Leu Glu Gly Ala Glu Leu Ser Val Leu Gln Leu 105110 115 aac acc aat gaa cgt ttg tgc gtc agg ttt gag ttt ctg tcc aaa ctg497 Asn Thr Asn Glu Arg Leu Cys Val Arg Phe Glu Phe Leu Ser Lys Leu 120125 130 135 agg cat cac cac agg cgg tgg cgt ttt acc ttc agc cac ttt gtggtt 545 Arg His His His Arg Arg Trp Arg Phe Thr Phe Ser His Phe Val Val140 145 150 gac cct gac cag gaa tat gag gtg acc gtt cac cac ctg ccc aagccc 593 Asp Pro Asp Gln Glu Tyr Glu Val Thr Val His His Leu Pro Lys Pro155 160 165 atc cct gat ggg gac cca aac cac cag tcc aag aat ttc ctt gtgcct 641 Ile Pro Asp Gly Asp Pro Asn His Gln Ser Lys Asn Phe Leu Val Pro170 175 180 gac tgt gag cac gcc agg atg aag gta acc acg cca tgc atg agctca 689 Asp Cys Glu His Ala Arg Met Lys Val Thr Thr Pro Cys Met Ser Ser185 190 195 ggc agc ctg tgg gac ccc aac atc acc gtg gag acc ctg gag gcccac 737 Gly Ser Leu Trp Asp Pro Asn Ile Thr Val Glu Thr Leu Glu Ala His200 205 210 215 cag ctg cgt gtg agc ttc acc ctg tgg aac gaa tct acc cattac cag 785 Gln Leu Arg Val Ser Phe Thr Leu Trp Asn Glu Ser Thr His TyrGln 220 225 230 atc ctg ctg acc agt ttt ccg cac atg gag aac cac agt tgcttt gag 833 Ile Leu Leu Thr Ser Phe Pro His Met Glu Asn His Ser Cys PheGlu 235 240 245 cac atg cac cac ata cct gcg ccc aga cca gaa gag ttc caccag cga 881 His Met His His Ile Pro Ala Pro Arg Pro Glu Glu Phe His GlnArg 250 255 260 tcc aac gtc aca ctc act cta cgc aac ctt aaa ggg tgc tgtcgc cac 929 Ser Asn Val Thr Leu Thr Leu Arg Asn Leu Lys Gly Cys Cys ArgHis 265 270 275 caa gtg cag atc cag ccc ttc ttc agc agc tgc ctc aat gactgc ctc 977 Gln Val Gln Ile Gln Pro Phe Phe Ser Ser Cys Leu Asn Asp CysLeu 280 285 290 295 aga cac tcc gcg act gtt tcc tgc cca gaa atg cca gacact cca gaa 1025 Arg His Ser Ala Thr Val Ser Cys Pro Glu Met Pro Asp ThrPro Glu 300 305 310 cca att ccg gac tac atg ccc ctg tgg gtg tac tgg ttcatc acg ggc 1073 Pro Ile Pro Asp Tyr Met Pro Leu Trp Val Tyr Trp Phe IleThr Gly 315 320 325 atc tcc atc ctg ctg gtg ggc tcc gtc atc ctg ctc atcgtc tgc atg 1121 Ile Ser Ile Leu Leu Val Gly Ser Val Ile Leu Leu Ile ValCys Met 330 335 340 acc tgg agg cta gct ggg cct gga agt gaa aaa tac agtgat gac acc 1169 Thr Trp Arg Leu Ala Gly Pro Gly Ser Glu Lys Tyr Ser AspAsp Thr 345 350 355 aaa tac acc gat ggc ctg cct gcg gct gac ctg atc ccccca ccg ctg 1217 Lys Tyr Thr Asp Gly Leu Pro Ala Ala Asp Leu Ile Pro ProPro Leu 360 365 370 375 aag ccc agg aag gtc tgg atc atc tac tca gcc gaccac ccc ctc tac 1265 Lys Pro Arg Lys Val Trp Ile Ile Tyr Ser Ala Asp HisPro Leu Tyr 380 385 390 gtg gac gtg gtc ctg aaa ttc gcc cag ttc ctg ctcacc gcc tgc ggc 1313 Val Asp Val Val Leu Lys Phe Ala Gln Phe Leu Leu ThrAla Cys Gly 395 400 405 acg gaa gtg gcc ctg gac ctg ctg gaa gag cag gccatc tcg gag gca 1361 Thr Glu Val Ala Leu Asp Leu Leu Glu Glu Gln Ala IleSer Glu Ala 410 415 420 gga gtc atg acc tgg gtg ggc cgt cag aag cag gagatg gtg gag agc 1409 Gly Val Met Thr Trp Val Gly Arg Gln Lys Gln Glu MetVal Glu Ser 425 430 435 aac tct aag atc atc gtc ctg tgc tcc cgc ggc acgcgc gcc aag tgg 1457 Asn Ser Lys Ile Ile Val Leu Cys Ser Arg Gly Thr ArgAla Lys Trp 440 445 450 455 cag gcg ctc ctg ggc cgg ggg gcg cct gtg cggctg cgc tgc gac cac 1505 Gln Ala Leu Leu Gly Arg Gly Ala Pro Val Arg LeuArg Cys Asp His 460 465 470 gga aag ccc gtg ggg gac ctg ttc act gca gccatg aac atg atc ctc 1553 Gly Lys Pro Val Gly Asp Leu Phe Thr Ala Ala MetAsn Met Ile Leu 475 480 485 ccg gac ttc aag agg cca gcc tgc ttc ggc acctac gta gtc tgc tac 1601 Pro Asp Phe Lys Arg Pro Ala Cys Phe Gly Thr TyrVal Val Cys Tyr 490 495 500 ttc agc gag gtc agc tgt gac ggc gac gtc cccgac ctg ttc ggc gcg 1649 Phe Ser Glu Val Ser Cys Asp Gly Asp Val Pro AspLeu Phe Gly Ala 505 510 515 gcg ccg cgg tac ccg ctc atg gac agg ttc gaggag gtg tac ttc cgc 1697 Ala Pro Arg Tyr Pro Leu Met Asp Arg Phe Glu GluVal Tyr Phe Arg 520 525 530 535 atc cag gac ctg gag atg ttc cag ccg ggccgc atg cac cgc gta ggg 1745 Ile Gln Asp Leu Glu Met Phe Gln Pro Gly ArgMet His Arg Val Gly 540 545 550 gag ctg tcg ggg gac aac tac ctg cgg agcccg ggc ggc agg cag ctc 1793 Glu Leu Ser Gly Asp Asn Tyr Leu Arg Ser ProGly Gly Arg Gln Leu 555 560 565 cgc gcc gcc ctg gac agg ttc cgg gac tggcag gtc cgc tgt ccc gac 1841 Arg Ala Ala Leu Asp Arg Phe Arg Asp Trp GlnVal Arg Cys Pro Asp 570 575 580 tgg ttc gaa tgt gag aac ctc tac tca gcagat gac cag gat gcc ccg 1889 Trp Phe Glu Cys Glu Asn Leu Tyr Ser Ala AspAsp Gln Asp Ala Pro 585 590 595 tcc ctg gac gaa gag gtg ttt gag gag ccactg ctg cct ccg gga acc 1937 Ser Leu Asp Glu Glu Val Phe Glu Glu Pro LeuLeu Pro Pro Gly Thr 600 605 610 615 ggc atc gtg aag cgg gcg ccc ctg gtgcgc gag cct ggc tcc cag gcc 1985 Gly Ile Val Lys Arg Ala Pro Leu Val ArgGlu Pro Gly Ser Gln Ala 620 625 630 tgc ctg gcc ata gac ccg ctg gtc ggggag gaa gga gga gca gca gtg 2033 Cys Leu Ala Ile Asp Pro Leu Val Gly GluGlu Gly Gly Ala Ala Val 635 640 645 gca aag ctg gaa cct cac ctg cag ccccgg ggt cag cca gcg ccg cag 2081 Ala Lys Leu Glu Pro His Leu Gln Pro ArgGly Gln Pro Ala Pro Gln 650 655 660 ccc ctc cac acc ctg gtg ctc gcc gcagag gag ggg gcc ctg gtg gcc 2129 Pro Leu His Thr Leu Val Leu Ala Ala GluGlu Gly Ala Leu Val Ala 665 670 675 gcg gtg gag cct ggg ccc ctg gct gacggt gcc gca gtc cgg ctg gca 2177 Ala Val Glu Pro Gly Pro Leu Ala Asp GlyAla Ala Val Arg Leu Ala 680 685 690 695 ctg gcg ggg gag ggc gag gcc tgcccg ctg ctg ggc agc ccg ggc gct 2225 Leu Ala Gly Glu Gly Glu Ala Cys ProLeu Leu Gly Ser Pro Gly Ala 700 705 710 ggg cga aat agc gtc ctc ttc ctcccc gtg gac ccc gag gac tcg ccc 2273 Gly Arg Asn Ser Val Leu Phe Leu ProVal Asp Pro Glu Asp Ser Pro 715 720 725 ctt ggc agc agc acc ccc atg gcgtct cct gac ctc ctt cca gag gac 2321 Leu Gly Ser Ser Thr Pro Met Ala SerPro Asp Leu Leu Pro Glu Asp 730 735 740 gtg agg gag cac ctc gaa ggc ttgatg ctc tcg ctc ttc gag cag agt 2369 Val Arg Glu His Leu Glu Gly Leu MetLeu Ser Leu Phe Glu Gln Ser 745 750 755 ctg agc tgc cag gcc cag ggg ggctgc agt aga ccc gcc atg gtc ctc 2417 Leu Ser Cys Gln Ala Gln Gly Gly CysSer Arg Pro Ala Met Val Leu 760 765 770 775 aca gac cca cac acg ccc tacgag gag gag cag cgg cag tca gtg cag 2465 Thr Asp Pro His Thr Pro Tyr GluGlu Glu Gln Arg Gln Ser Val Gln 780 785 790 tct gac cag ggc tac atc tccagg agc tcc ccg cag ccc ccc gag gga 2513 Ser Asp Gln Gly Tyr Ile Ser ArgSer Ser Pro Gln Pro Pro Glu Gly 795 800 805 ctc acg gaa atg gag gaa gaggag gaa gag gag cag gac cca ggg aag 2561 Leu Thr Glu Met Glu Glu Glu GluGlu Glu Glu Gln Asp Pro Gly Lys 810 815 820 ccg gcc ctg cca ctc tct cccgag gac ctg gag agc ctg agg agc ctc 2609 Pro Ala Leu Pro Leu Ser Pro GluAsp Leu Glu Ser Leu Arg Ser Leu 825 830 835 cag cgg cag ctg ctt ttc cgccag ctg cag aag aac tcg ggc tgg gac 2657 Gln Arg Gln Leu Leu Phe Arg GlnLeu Gln Lys Asn Ser Gly Trp Asp 840 845 850 855 acg atg ggg tca gag tcagag ggg ccc agt gca tga gggcggctcc 2703 Thr Met Gly Ser Glu Ser Glu GlyPro Ser Ala 860 865 ccagggaccg cccagatccc agctttgaga gaggagtgtgtgtgcacgta ttcatctgtg 2763 tgtacatgtc tgcatgtgta tatgttcgtg tgtgaaatgtaggctttaaa atgtaaatgt 2823 ctggatttta atcccaggca tccctcctaa cttttctttgtgcagcggtc tggttatcgt 2883 ctatccccag gggaatccac acagcccgct cccaggagctaatggtagag cgtccttgag 2943 gctccattat tcgttcattc agcatttatt gtgcacctactatgtggcgg gcatttggga 3003 taccaagata aattgcatgc ggcatggccc cagccatgaaggaacttaac cgctagtgcc 3063 gaggacacgt taaacgaaca ggatgggccg ggcacggtggctcacgcctg taatcccagc 3123 acactgggag gccgaggcag gtggatcact ctgaggtcaggagtttgagc cagcctggcc 3183 aacatggtga aaccccggaa ttcgagctcg gtacccgggg3223 4 866 PRT Homo sapiens 4 Met Gly Ala Ala Arg Ser Pro Pro Ser AlaVal Pro Gly Pro Leu Leu 1 5 10 15 Gly Leu Leu Leu Leu Leu Leu Gly ValLeu Ala Pro Gly Gly Ala Ser 20 25 30 Leu Arg Leu Leu Asp His Arg Ala LeuVal Cys Ser Gln Pro Gly Leu 35 40 45 Asn Cys Thr Val Lys Asn Ser Thr CysLeu Asp Asp Ser Trp Ile His 50 55 60 Pro Arg Asn Leu Thr Pro Ser Ser ProLys Asp Leu Gln Ile Gln Leu 65 70 75 80 His Phe Ala His Thr Gln Gln GlyAsp Leu Phe Pro Val Ala His Ile 85 90 95 Glu Trp Thr Leu Gln Thr Asp AlaSer Ile Leu Tyr Leu Glu Gly Ala 100 105 110 Glu Leu Ser Val Leu Gln LeuAsn Thr Asn Glu Arg Leu Cys Val Arg 115 120 125 Phe Glu Phe Leu Ser LysLeu Arg His His His Arg Arg Trp Arg Phe 130 135 140 Thr Phe Ser His PheVal Val Asp Pro Asp Gln Glu Tyr Glu Val Thr 145 150 155 160 Val His HisLeu Pro Lys Pro Ile Pro Asp Gly Asp Pro Asn His Gln 165 170 175 Ser LysAsn Phe Leu Val Pro Asp Cys Glu His Ala Arg Met Lys Val 180 185 190 ThrThr Pro Cys Met Ser Ser Gly Ser Leu Trp Asp Pro Asn Ile Thr 195 200 205Val Glu Thr Leu Glu Ala His Gln Leu Arg Val Ser Phe Thr Leu Trp 210 215220 Asn Glu Ser Thr His Tyr Gln Ile Leu Leu Thr Ser Phe Pro His Met 225230 235 240 Glu Asn His Ser Cys Phe Glu His Met His His Ile Pro Ala ProArg 245 250 255 Pro Glu Glu Phe His Gln Arg Ser Asn Val Thr Leu Thr LeuArg Asn 260 265 270 Leu Lys Gly Cys Cys Arg His Gln Val Gln Ile Gln ProPhe Phe Ser 275 280 285 Ser Cys Leu Asn Asp Cys Leu Arg His Ser Ala ThrVal Ser Cys Pro 290 295 300 Glu Met Pro Asp Thr Pro Glu Pro Ile Pro AspTyr Met Pro Leu Trp 305 310 315 320 Val Tyr Trp Phe Ile Thr Gly Ile SerIle Leu Leu Val Gly Ser Val 325 330 335 Ile Leu Leu Ile Val Cys Met ThrTrp Arg Leu Ala Gly Pro Gly Ser 340 345 350 Glu Lys Tyr Ser Asp Asp ThrLys Tyr Thr Asp Gly Leu Pro Ala Ala 355 360 365 Asp Leu Ile Pro Pro ProLeu Lys Pro Arg Lys Val Trp Ile Ile Tyr 370 375 380 Ser Ala Asp His ProLeu Tyr Val Asp Val Val Leu Lys Phe Ala Gln 385 390 395 400 Phe Leu LeuThr Ala Cys Gly Thr Glu Val Ala Leu Asp Leu Leu Glu 405 410 415 Glu GlnAla Ile Ser Glu Ala Gly Val Met Thr Trp Val Gly Arg Gln 420 425 430 LysGln Glu Met Val Glu Ser Asn Ser Lys Ile Ile Val Leu Cys Ser 435 440 445Arg Gly Thr Arg Ala Lys Trp Gln Ala Leu Leu Gly Arg Gly Ala Pro 450 455460 Val Arg Leu Arg Cys Asp His Gly Lys Pro Val Gly Asp Leu Phe Thr 465470 475 480 Ala Ala Met Asn Met Ile Leu Pro Asp Phe Lys Arg Pro Ala CysPhe 485 490 495 Gly Thr Tyr Val Val Cys Tyr Phe Ser Glu Val Ser Cys AspGly Asp 500 505 510 Val Pro Asp Leu Phe Gly Ala Ala Pro Arg Tyr Pro LeuMet Asp Arg 515 520 525 Phe Glu Glu Val Tyr Phe Arg Ile Gln Asp Leu GluMet Phe Gln Pro 530 535 540 Gly Arg Met His Arg Val Gly Glu Leu Ser GlyAsp Asn Tyr Leu Arg 545 550 555 560 Ser Pro Gly Gly Arg Gln Leu Arg AlaAla Leu Asp Arg Phe Arg Asp 565 570 575 Trp Gln Val Arg Cys Pro Asp TrpPhe Glu Cys Glu Asn Leu Tyr Ser 580 585 590 Ala Asp Asp Gln Asp Ala ProSer Leu Asp Glu Glu Val Phe Glu Glu 595 600 605 Pro Leu Leu Pro Pro GlyThr Gly Ile Val Lys Arg Ala Pro Leu Val 610 615 620 Arg Glu Pro Gly SerGln Ala Cys Leu Ala Ile Asp Pro Leu Val Gly 625 630 635 640 Glu Glu GlyGly Ala Ala Val Ala Lys Leu Glu Pro His Leu Gln Pro 645 650 655 Arg GlyGln Pro Ala Pro Gln Pro Leu His Thr Leu Val Leu Ala Ala 660 665 670 GluGlu Gly Ala Leu Val Ala Ala Val Glu Pro Gly Pro Leu Ala Asp 675 680 685Gly Ala Ala Val Arg Leu Ala Leu Ala Gly Glu Gly Glu Ala Cys Pro 690 695700 Leu Leu Gly Ser Pro Gly Ala Gly Arg Asn Ser Val Leu Phe Leu Pro 705710 715 720 Val Asp Pro Glu Asp Ser Pro Leu Gly Ser Ser Thr Pro Met AlaSer 725 730 735 Pro Asp Leu Leu Pro Glu Asp Val Arg Glu His Leu Glu GlyLeu Met 740 745 750 Leu Ser Leu Phe Glu Gln Ser Leu Ser Cys Gln Ala GlnGly Gly Cys 755 760 765 Ser Arg Pro Ala Met Val Leu Thr Asp Pro His ThrPro Tyr Glu Glu 770 775 780 Glu Gln Arg Gln Ser Val Gln Ser Asp Gln GlyTyr Ile Ser Arg Ser 785 790 795 800 Ser Pro Gln Pro Pro Glu Gly Leu ThrGlu Met Glu Glu Glu Glu Glu 805 810 815 Glu Glu Gln Asp Pro Gly Lys ProAla Leu Pro Leu Ser Pro Glu Asp 820 825 830 Leu Glu Ser Leu Arg Ser LeuGln Arg Gln Leu Leu Phe Arg Gln Leu 835 840 845 Gln Lys Asn Ser Gly TrpAsp Thr Met Gly Ser Glu Ser Glu Gly Pro 850 855 860 Ser Ala 865 5 1626DNA Homo sapeins CDS (1)..(1626) 5 atg aat tgt aga gaa tta ccc ttg accctt tgg gtg ctt ata tct gta 48 Met Asn Cys Arg Glu Leu Pro Leu Thr LeuTrp Val Leu Ile Ser Val 1 5 10 15 agc act gca gaa tct tgt act tca cgtccc cac att act gtg gtt gaa 96 Ser Thr Ala Glu Ser Cys Thr Ser Arg ProHis Ile Thr Val Val Glu 20 25 30 ggg gaa cct ttc tat ctg aaa cat tgc tcgtgt tca ctt gca cat gag 144 Gly Glu Pro Phe Tyr Leu Lys His Cys Ser CysSer Leu Ala His Glu 35 40 45 att gaa aca acc acc aaa agc tgg tac aaa agcagt gga tca cag gaa 192 Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys Ser SerGly Ser Gln Glu 50 55 60 cat gtg gag ctg aac cca agg agt tcc tcg aga attgct ttg cat gat 240 His Val Glu Leu Asn Pro Arg Ser Ser Ser Arg Ile AlaLeu His Asp 65 70 75 80 tgt gtt ttg gag ttt tgg cca gtt gag ttg aat gacaca gga tct tac 288 Cys Val Leu Glu Phe Trp Pro Val Glu Leu Asn Asp ThrGly Ser Tyr 85 90 95 ttt ttc caa atg aaa aat tat act cag aaa tgg aaa ttaaat gtc atc 336 Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu AsnVal Ile 100 105 110 aga aga aat aaa cac agc tgt ttc act gaa aga caa gtaact agt aaa 384 Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg Gln Val ThrSer Lys 115 120 125 att gtg gaa gtt aaa aaa ttt ttt cag ata acc tgt gaaaac agt tac 432 Ile Val Glu Val Lys Lys Phe Phe Gln Ile Thr Cys Glu AsnSer Tyr 130 135 140 tat caa aca ctg gtc aac agc aca tca ttg tat aag aactgt aaa aag 480 Tyr Gln Thr Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn CysLys Lys 145 150 155 160 cta cta ctg gag aac aat aaa aac cca acg ata aagaag aac gcc gag 528 Leu Leu Leu Glu Asn Asn Lys Asn Pro Thr Ile Lys LysAsn Ala Glu 165 170 175 ttt gaa gat cag ggg tat tac tcc tgc gtg cat ttcctt cat cat aat 576 Phe Glu Asp Gln Gly Tyr Tyr Ser Cys Val His Phe LeuHis His Asn 180 185 190 gga aaa cta ttt aat atc acc aaa acc ttc aat ataaca ata gtg gaa 624 Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe Asn Ile ThrIle Val Glu 195 200 205 gat cgc agt aat ata gtt ccg gtt ctt ctt gga ccaaag ctt aac cat 672 Asp Arg Ser Asn Ile Val Pro Val Leu Leu Gly Pro LysLeu Asn His 210 215 220 gtt gca gtg gaa tta gga aaa aac gta agg ctc aactgc tct gct ttg 720 Val Ala Val Glu Leu Gly Lys Asn Val Arg Leu Asn CysSer Ala Leu 225 230 235 240 ctg aat gaa gag gat gta att tat tgg atg tttggg gaa gaa aat gga 768 Leu Asn Glu Glu Asp Val Ile Tyr Trp Met Phe GlyGlu Glu Asn Gly 245 250 255 tcg gat cct aat ata cat gaa gag aaa gaa atgaga att atg act cca 816 Ser Asp Pro Asn Ile His Glu Glu Lys Glu Met ArgIle Met Thr Pro 260 265 270 gaa ggc aaa tgg cat gct tca aaa gta ttg agaatt gaa aat att ggt 864 Glu Gly Lys Trp His Ala Ser Lys Val Leu Arg IleGlu Asn Ile Gly 275 280 285 gaa agc aat cta aat gtt tta tat aat tgc actgtg gcc agc acg gga 912 Glu Ser Asn Leu Asn Val Leu Tyr Asn Cys Thr ValAla Ser Thr Gly 290 295 300 ggc aca gac acc aaa agc ttc atc ttg gtg agaaaa gca gac atg gct 960 Gly Thr Asp Thr Lys Ser Phe Ile Leu Val Arg LysAla Asp Met Ala 305 310 315 320 gat atc cca ggc cac gtc ttc aca aga ggaatg atc ata gct gtt ttg 1008 Asp Ile Pro Gly His Val Phe Thr Arg Gly MetIle Ile Ala Val Leu 325 330 335 atc ttg gtg gca gta gtg tgc cta gtg actgtg tgt gtc att tat aga 1056 Ile Leu Val Ala Val Val Cys Leu Val Thr ValCys Val Ile Tyr Arg 340 345 350 gtt gac ttg gtt cta ttt tat aga cat ttaacg aga aga gat gaa aca 1104 Val Asp Leu Val Leu Phe Tyr Arg His Leu ThrArg Arg Asp Glu Thr 355 360 365 tta aca gat gga aaa aca tat gat gct tttgtg tct tac cta aaa gaa 1152 Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe ValSer Tyr Leu Lys Glu 370 375 380 tgc cga cct gaa aat gga gag gag cac accttt gct gtg gag att ttg 1200 Cys Arg Pro Glu Asn Gly Glu Glu His Thr PheAla Val Glu Ile Leu 385 390 395 400 ccc agg gtg ttg gag aaa cat ttt gggtat aag tta tgc ata ttt gaa 1248 Pro Arg Val Leu Glu Lys His Phe Gly TyrLys Leu Cys Ile Phe Glu 405 410 415 agg gat gta gtg cct gga gga gct gttgtt gat gaa atc cac tca ctg 1296 Arg Asp Val Val Pro Gly Gly Ala Val ValAsp Glu Ile His Ser Leu 420 425 430 ata gag aaa agc cga aga cta atc attgtc cta agt aaa agt tat atg 1344 Ile Glu Lys Ser Arg Arg Leu Ile Ile ValLeu Ser Lys Ser Tyr Met 435 440 445 tct aat gag gtc agg tat gaa ctt gaaagt gga ctc cat gaa gca ttg 1392 Ser Asn Glu Val Arg Tyr Glu Leu Glu SerGly Leu His Glu Ala Leu 450 455 460 gtg gaa aga aaa att aaa ata atc ttaatt gaa ttt aca cct gtt act 1440 Val Glu Arg Lys Ile Lys Ile Ile Leu IleGlu Phe Thr Pro Val Thr 465 470 475 480 gac ttc aca ttc ttg ccc caa tcacta aag ctt ttg aaa tct cac aga 1488 Asp Phe Thr Phe Leu Pro Gln Ser LeuLys Leu Leu Lys Ser His Arg 485 490 495 gtt ctg aag tgg aag gcc gat aaatct ctt tct tat aac tca agg ttc 1536 Val Leu Lys Trp Lys Ala Asp Lys SerLeu Ser Tyr Asn Ser Arg Phe 500 505 510 tgg aag aac ctt ctt tac tta atgcct gca aaa aca gtc aag cca ggt 1584 Trp Lys Asn Leu Leu Tyr Leu Met ProAla Lys Thr Val Lys Pro Gly 515 520 525 aga gac gaa ccg gaa gtc ttg cctgtt ctt tcc gag tct taa 1626 Arg Asp Glu Pro Glu Val Leu Pro Val Leu SerGlu Ser 530 535 540 6 541 PRT Homo sapeins 6 Met Asn Cys Arg Glu Leu ProLeu Thr Leu Trp Val Leu Ile Ser Val 1 5 10 15 Ser Thr Ala Glu Ser CysThr Ser Arg Pro His Ile Thr Val Val Glu 20 25 30 Gly Glu Pro Phe Tyr LeuLys His Cys Ser Cys Ser Leu Ala His Glu 35 40 45 Ile Glu Thr Thr Thr LysSer Trp Tyr Lys Ser Ser Gly Ser Gln Glu 50 55 60 His Val Glu Leu Asn ProArg Ser Ser Ser Arg Ile Ala Leu His Asp 65 70 75 80 Cys Val Leu Glu PheTrp Pro Val Glu Leu Asn Asp Thr Gly Ser Tyr 85 90 95 Phe Phe Gln Met LysAsn Tyr Thr Gln Lys Trp Lys Leu Asn Val Ile 100 105 110 Arg Arg Asn LysHis Ser Cys Phe Thr Glu Arg Gln Val Thr Ser Lys 115 120 125 Ile Val GluVal Lys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser Tyr 130 135 140 Tyr GlnThr Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys 145 150 155 160Leu Leu Leu Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu 165 170175 Phe Glu Asp Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His His Asn 180185 190 Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val Glu195 200 205 Asp Arg Ser Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu AsnHis 210 215 220 Val Ala Val Glu Leu Gly Lys Asn Val Arg Leu Asn Cys SerAla Leu 225 230 235 240 Leu Asn Glu Glu Asp Val Ile Tyr Trp Met Phe GlyGlu Glu Asn Gly 245 250 255 Ser Asp Pro Asn Ile His Glu Glu Lys Glu MetArg Ile Met Thr Pro 260 265 270 Glu Gly Lys Trp His Ala Ser Lys Val LeuArg Ile Glu Asn Ile Gly 275 280 285 Glu Ser Asn Leu Asn Val Leu Tyr AsnCys Thr Val Ala Ser Thr Gly 290 295 300 Gly Thr Asp Thr Lys Ser Phe IleLeu Val Arg Lys Ala Asp Met Ala 305 310 315 320 Asp Ile Pro Gly His ValPhe Thr Arg Gly Met Ile Ile Ala Val Leu 325 330 335 Ile Leu Val Ala ValVal Cys Leu Val Thr Val Cys Val Ile Tyr Arg 340 345 350 Val Asp Leu ValLeu Phe Tyr Arg His Leu Thr Arg Arg Asp Glu Thr 355 360 365 Leu Thr AspGly Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu Lys Glu 370 375 380 Cys ArgPro Glu Asn Gly Glu Glu His Thr Phe Ala Val Glu Ile Leu 385 390 395 400Pro Arg Val Leu Glu Lys His Phe Gly Tyr Lys Leu Cys Ile Phe Glu 405 410415 Arg Asp Val Val Pro Gly Gly Ala Val Val Asp Glu Ile His Ser Leu 420425 430 Ile Glu Lys Ser Arg Arg Leu Ile Ile Val Leu Ser Lys Ser Tyr Met435 440 445 Ser Asn Glu Val Arg Tyr Glu Leu Glu Ser Gly Leu His Glu AlaLeu 450 455 460 Val Glu Arg Lys Ile Lys Ile Ile Leu Ile Glu Phe Thr ProVal Thr 465 470 475 480 Asp Phe Thr Phe Leu Pro Gln Ser Leu Lys Leu LeuLys Ser His Arg 485 490 495 Val Leu Lys Trp Lys Ala Asp Lys Ser Leu SerTyr Asn Ser Arg Phe 500 505 510 Trp Lys Asn Leu Leu Tyr Leu Met Pro AlaLys Thr Val Lys Pro Gly 515 520 525 Arg Asp Glu Pro Glu Val Leu Pro ValLeu Ser Glu Ser 530 535 540 7 2681 DNA Homo sapiens CDS (484)..(2283) 7ctctctggat aggaagaaat atagtagaac cctttgaaaa tggatatttt cacatatttt 60cgttcagata caaaagctgg cagttactga aataaggact tgaagttcct tcctcttttt 120ttatgtctta agagcaggaa ataaagagac agctgaaggt gtagccttga ccaactgaaa 180gggaaatctt catcctctga aaaaacatat gtgattctca aaaaacgcat ctggaaaatt 240gataaagaag cgattctgta gattctccca gcgctgttgg gctctcaatt ccttctgtga 300aggacaacat atggtgatgg ggaaatcaga agctttgaga ccctctacac ctggatatga 360atcccccttc taatacttac cagaaatgaa ggggatactc agggcagagt tctgaatctc 420aaaacactct actctggcaa aggaatgaag ttattggagt gatgacagga acacgggaga 480aca atg ctc tgt ttg ggc tgg ata ttt ctt tgg ctt gtt gca gga gag 528 MetLeu Cys Leu Gly Trp Ile Phe Leu Trp Leu Val Ala Gly Glu 1 5 10 15 cgaatt aaa gga ttt aat att tca ggt tgt tcc aca aaa aaa ctc ctt 576 Arg IleLys Gly Phe Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu 20 25 30 tgg acatat tct aca agg agt gaa gag gaa ttt gtc tta ttt tgt gat 624 Trp Thr TyrSer Thr Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp 35 40 45 tta cca gagcca cag aaa tca cat ttc tgc cac aga aat cga ctc tca 672 Leu Pro Glu ProGln Lys Ser His Phe Cys His Arg Asn Arg Leu Ser 50 55 60 cca aaa caa gtccct gag cac ctg ccc ttc atg ggt agt aac gac cta 720 Pro Lys Gln Val ProGlu His Leu Pro Phe Met Gly Ser Asn Asp Leu 65 70 75 tct gat gtc caa tggtac caa caa cct tcg aat gga gat cca tta gag 768 Ser Asp Val Gln Trp TyrGln Gln Pro Ser Asn Gly Asp Pro Leu Glu 80 85 90 95 gac att agg aaa agctat cct cac atc att cag gac aaa tgt acc ctt 816 Asp Ile Arg Lys Ser TyrPro His Ile Ile Gln Asp Lys Cys Thr Leu 100 105 110 cac ttt ttg acc ccaggg gtg aat aat tct ggg tca tat att tgt aga 864 His Phe Leu Thr Pro GlyVal Asn Asn Ser Gly Ser Tyr Ile Cys Arg 115 120 125 ccc aag atg att aagagc ccc tat gat gta gcc tgt tgt gtc aag atg 912 Pro Lys Met Ile Lys SerPro Tyr Asp Val Ala Cys Cys Val Lys Met 130 135 140 att tta gaa gtt aagccc cag aca aat gca tcc tgt gag tat tcc gca 960 Ile Leu Glu Val Lys ProGln Thr Asn Ala Ser Cys Glu Tyr Ser Ala 145 150 155 tca cat aag caa gaccta ctt ctt ggg agc act ggc tct att tct tgc 1008 Ser His Lys Gln Asp LeuLeu Leu Gly Ser Thr Gly Ser Ile Ser Cys 160 165 170 175 ccc agt ctc agctgc caa agt gat gca caa agt cca gcg gta acc tgg 1056 Pro Ser Leu Ser CysGln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp 180 185 190 tac aag aat ggaaaa ctc ctc tct gtg gaa agg agc aac cga atc gta 1104 Tyr Lys Asn Gly LysLeu Leu Ser Val Glu Arg Ser Asn Arg Ile Val 195 200 205 gtg gat gaa gtttat gac tat cac cag ggc aca tat gta tgt gat tac 1152 Val Asp Glu Val TyrAsp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr 210 215 220 act cag tcg gatact gtg agt tcg tgg aca gtc aga gct gtt gtt caa 1200 Thr Gln Ser Asp ThrVal Ser Ser Trp Thr Val Arg Ala Val Val Gln 225 230 235 gtg aga acc attgtg gga gac act aaa ctc aaa cca gat att ctg gat 1248 Val Arg Thr Ile ValGly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp 240 245 250 255 cct gtc gaggac aca ctg gaa gta gaa ctt gga aag cct tta act att 1296 Pro Val Glu AspThr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile 260 265 270 agc tgc aaagca cga ttt ggc ttt gaa agg gtc ttt aac cct gtc ata 1344 Ser Cys Lys AlaArg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile 275 280 285 aaa tgg tacatc aaa gat tct gac cta gag tgg gaa gtc tca gta cct 1392 Lys Trp Tyr IleLys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro 290 295 300 gag gcg aaaagt att aaa tcc act tta aag gat gaa atc att gag cgt 1440 Glu Ala Lys SerIle Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg 305 310 315 aat atc atcttg gaa aaa gtc act cag cgt gat ctt cgc agg aag ttt 1488 Asn Ile Ile LeuGlu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe 320 325 330 335 gtt tgcttt gtc cag aac tcc att gga aac aca acc cag tcc gtc caa 1536 Val Cys PheVal Gln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln 340 345 350 ctg aaagaa aag aga gga gtg gtg ctc ctg tac atc ctg ctt ggc acc 1584 Leu Lys GluLys Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr 355 360 365 atc gggacc ctg gtg gcc gtg ctg gcg gcg agt gcc ctc ctc tac agg 1632 Ile Gly ThrLeu Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg 370 375 380 cac tggatt gaa ata gtg ctg ctg tac cgg acc tac cag agc aag gat 1680 His Trp IleGlu Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp 385 390 395 cag acgctt ggg gat aaa aag gat ttt gat gct ttc gta tcc tat gca 1728 Gln Thr LeuGly Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala 400 405 410 415 aaatgg agc tct ttt cca agt gag gcc act tca tct ctg agt gaa gaa 1776 Lys TrpSer Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu 420 425 430 cacttg gcc ctg agc cta ttt cct gat gtt tta gaa aac aaa tat gga 1824 His LeuAla Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly 435 440 445 tatagc ctg tgt ttg ctt gaa aga gat gtg gct cca gga gga gtg tat 1872 Tyr SerLeu Cys Leu Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr 450 455 460 gcagaa gac att gtg agc att att aag aga agc aga aga gga ata ttt 1920 Ala GluAsp Ile Val Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe 465 470 475 atcttg agc ccc aac tat gtc aat gga ccc agt atc ttt gaa cta caa 1968 Ile LeuSer Pro Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln 480 485 490 495gca gca gtg aat ctt gcc ttg gat gat caa aca ctg aaa ctc att tta 2016 AlaAla Val Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu 500 505 510att aag ttc tgt tac ttc caa gag cca gag tct cta cct cat ctc gtg 2064 IleLys Phe Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val 515 520 525aaa aaa gct ctc agg gtt ttg ccc aca gtt act tgg aga ggc tta aaa 2112 LysLys Ala Leu Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys 530 535 540tca gtt cct ccc aat tct agg ttc tgg gcc aaa atg cgc tac cac atg 2160 SerVal Pro Pro Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met 545 550 555cct gtg aaa aac tct cag gga ttc acg tgg aac cag ctc aga att acc 2208 ProVal Lys Asn Ser Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr 560 565 570575 tct agg att ttt cag tgg aaa gga ctc agt aga aca gaa acc act ggg 2256Ser Arg Ile Phe Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly 580 585590 agg agc tcc cag cct aag gaa tgg tga aatgagccct ggagccccct 2303 ArgSer Ser Gln Pro Lys Glu Trp 595 ccagtccagt ccctgggata gagatgttgctggacagaac tcacagctct gtgtgtgtgt 2363 gttcaggctg ataggaaatt caaagagtctcctgccagca ccaagcaagc ttgatggaca 2423 atggaatggg attgagactg tggtttagagcctttgattt cctggactgg acagacggcg 2483 agtgaattct ctagaccttg ggtactttcagtacacaaca cccctaagat ttcccagtgg 2543 tccgagcaga atcagaaaat acagctacttctgccttatg gctagggaac tgtcatgtct 2603 accatgtatt gtacatatga ctttatgtatacttgcaatc aaataaatat tattttatta 2663 gaaaaaaaac cggaattc 2681 8 599 PRTHomo sapiens 8 Met Leu Cys Leu Gly Trp Ile Phe Leu Trp Leu Val Ala GlyGlu Arg 1 5 10 15 Ile Lys Gly Phe Asn Ile Ser Gly Cys Ser Thr Lys LysLeu Leu Trp 20 25 30 Thr Tyr Ser Thr Arg Ser Glu Glu Glu Phe Val Leu PheCys Asp Leu 35 40 45 Pro Glu Pro Gln Lys Ser His Phe Cys His Arg Asn ArgLeu Ser Pro 50 55 60 Lys Gln Val Pro Glu His Leu Pro Phe Met Gly Ser AsnAsp Leu Ser 65 70 75 80 Asp Val Gln Trp Tyr Gln Gln Pro Ser Asn Gly AspPro Leu Glu Asp 85 90 95 Ile Arg Lys Ser Tyr Pro His Ile Ile Gln Asp LysCys Thr Leu His 100 105 110 Phe Leu Thr Pro Gly Val Asn Asn Ser Gly SerTyr Ile Cys Arg Pro 115 120 125 Lys Met Ile Lys Ser Pro Tyr Asp Val AlaCys Cys Val Lys Met Ile 130 135 140 Leu Glu Val Lys Pro Gln Thr Asn AlaSer Cys Glu Tyr Ser Ala Ser 145 150 155 160 His Lys Gln Asp Leu Leu LeuGly Ser Thr Gly Ser Ile Ser Cys Pro 165 170 175 Ser Leu Ser Cys Gln SerAsp Ala Gln Ser Pro Ala Val Thr Trp Tyr 180 185 190 Lys Asn Gly Lys LeuLeu Ser Val Glu Arg Ser Asn Arg Ile Val Val 195 200 205 Asp Glu Val TyrAsp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr 210 215 220 Gln Ser AspThr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val 225 230 235 240 ArgThr Ile Val Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp Pro 245 250 255Val Glu Asp Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile Ser 260 265270 Cys Lys Ala Arg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys 275280 285 Trp Tyr Ile Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu290 295 300 Ala Lys Ser Ile Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu ArgAsn 305 310 315 320 Ile Ile Leu Glu Lys Val Thr Gln Arg Asp Leu Arg ArgLys Phe Val 325 330 335 Cys Phe Val Gln Asn Ser Ile Gly Asn Thr Thr GlnSer Val Gln Leu 340 345 350 Lys Glu Lys Arg Gly Val Val Leu Leu Tyr IleLeu Leu Gly Thr Ile 355 360 365 Gly Thr Leu Val Ala Val Leu Ala Ala SerAla Leu Leu Tyr Arg His 370 375 380 Trp Ile Glu Ile Val Leu Leu Tyr ArgThr Tyr Gln Ser Lys Asp Gln 385 390 395 400 Thr Leu Gly Asp Lys Lys AspPhe Asp Ala Phe Val Ser Tyr Ala Lys 405 410 415 Trp Ser Ser Phe Pro SerGlu Ala Thr Ser Ser Leu Ser Glu Glu His 420 425 430 Leu Ala Leu Ser LeuPhe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr 435 440 445 Ser Leu Cys LeuLeu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala 450 455 460 Glu Asp IleVal Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile 465 470 475 480 LeuSer Pro Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala 485 490 495Ala Val Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile 500 505510 Lys Phe Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys 515520 525 Lys Ala Leu Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser530 535 540 Val Pro Pro Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His MetPro 545 550 555 560 Val Lys Asn Ser Gln Gly Phe Thr Trp Asn Gln Leu ArgIle Thr Ser 565 570 575 Arg Ile Phe Gln Trp Lys Gly Leu Ser Arg Thr GluThr Thr Gly Arg 580 585 590 Ser Ser Gln Pro Lys Glu Trp 595 9 644 DNAHomo sapiens CDS (66)..(599) 9 gagaagagga cgttgtcaca gataaagagccaggctcacc agctcctgac gcatgcatca 60 tgacc atg aga cac aac tgg aca ccagac ctc agc cct ttg tgg gtc ctg 110 Met Arg His Asn Trp Thr Pro Asp LeuSer Pro Leu Trp Val Leu 1 5 10 15 ctc ctg tgt gcc cac gtc gtc act ctcctg gtc aga gcc aca cct gtc 158 Leu Leu Cys Ala His Val Val Thr Leu LeuVal Arg Ala Thr Pro Val 20 25 30 tcg cag acc acc aca gct gcc act gcc tcagtt aga agc aca aag gac 206 Ser Gln Thr Thr Thr Ala Ala Thr Ala Ser ValArg Ser Thr Lys Asp 35 40 45 ccc tgc ccc tcc cag ccc cca gtg ttc cca gcagct aag cag tgt cca 254 Pro Cys Pro Ser Gln Pro Pro Val Phe Pro Ala AlaLys Gln Cys Pro 50 55 60 gca ttg gaa gtg acc tgg cca gag gtg gaa gtg ccactg aat gga acg 302 Ala Leu Glu Val Thr Trp Pro Glu Val Glu Val Pro LeuAsn Gly Thr 65 70 75 ctg agc tta tcc tgt gtg gcc tgc agc cgc ttc ccc aacttc agc atc 350 Leu Ser Leu Ser Cys Val Ala Cys Ser Arg Phe Pro Asn PheSer Ile 80 85 90 95 ctc tac tgg ctg ggc aat ggt tcc ttc att gag cac ctccca ggc cga 398 Leu Tyr Trp Leu Gly Asn Gly Ser Phe Ile Glu His Leu ProGly Arg 100 105 110 ctg tgg gag ggg agc acc agc cgg gaa cgt ggg agc acaggt acg cag 446 Leu Trp Glu Gly Ser Thr Ser Arg Glu Arg Gly Ser Thr GlyThr Gln 115 120 125 ctg tgc aag gcc ttg gtg ctg gag cag ctg acc cct gccctg cac agc 494 Leu Cys Lys Ala Leu Val Leu Glu Gln Leu Thr Pro Ala LeuHis Ser 130 135 140 acc aac ttc tcc tgt gtg ctc gtg gac cct gaa cag gttgtc cag cgt 542 Thr Asn Phe Ser Cys Val Leu Val Asp Pro Glu Gln Val ValGln Arg 145 150 155 cac gtc gtc ctg gcc cag ctc tgg gct ggg ctg agg gcaacc ttg ccc 590 His Val Val Leu Ala Gln Leu Trp Ala Gly Leu Arg Ala ThrLeu Pro 160 165 170 175 ccc acc caa gaagccctgc cctccagcca cagcagtccacagcagcagg gttaa 644 Pro Thr Gln 10 178 PRT Homo sapiens 10 Met Arg HisAsn Trp Thr Pro Asp Leu Ser Pro Leu Trp Val Leu Leu 1 5 10 15 Leu CysAla His Val Val Thr Leu Leu Val Arg Ala Thr Pro Val Ser 20 25 30 Gln ThrThr Thr Ala Ala Thr Ala Ser Val Arg Ser Thr Lys Asp Pro 35 40 45 Cys ProSer Gln Pro Pro Val Phe Pro Ala Ala Lys Gln Cys Pro Ala 50 55 60 Leu GluVal Thr Trp Pro Glu Val Glu Val Pro Leu Asn Gly Thr Leu 65 70 75 80 SerLeu Ser Cys Val Ala Cys Ser Arg Phe Pro Asn Phe Ser Ile Leu 85 90 95 TyrTrp Leu Gly Asn Gly Ser Phe Ile Glu His Leu Pro Gly Arg Leu 100 105 110Trp Glu Gly Ser Thr Ser Arg Glu Arg Gly Ser Thr Gly Thr Gln Leu 115 120125 Cys Lys Ala Leu Val Leu Glu Gln Leu Thr Pro Ala Leu His Ser Thr 130135 140 Asn Phe Ser Cys Val Leu Val Asp Pro Glu Gln Val Val Gln Arg His145 150 155 160 Val Val Leu Ala Gln Leu Trp Ala Gly Leu Arg Ala Thr LeuPro Pro 165 170 175 Thr Gln 11 422 PRT Homo sapiens 11 Met Arg His AsnTrp Thr Pro Asp Leu Ser Pro Leu Trp Val Leu Leu 1 5 10 15 Leu Cys AlaHis Val Val Thr Leu Leu Val Arg Ala Thr Pro Val Ser 20 25 30 Gln Thr ThrThr Ala Ala Thr Ala Ser Val Arg Ser Thr Lys Asp Pro 35 40 45 Cys Pro SerGln Pro Pro Val Phe Pro Ala Ala Lys Gln Cys Pro Ala 50 55 60 Leu Glu ValThr Trp Pro Glu Val Glu Val Pro Leu Asn Gly Thr Leu 65 70 75 80 Ser LeuSer Cys Val Ala Cys Ser Arg Phe Pro Asn Phe Ser Ile Leu 85 90 95 Tyr TrpLeu Gly Asn Gly Ser Phe Ile Glu His Leu Pro Gly Arg Leu 100 105 110 TrpGlu Gly Ser Thr Ser Arg Glu Arg Gly Ser Thr Gly Thr Gln Leu 115 120 125Cys Lys Ala Leu Val Leu Glu Gln Leu Thr Pro Ala Leu His Ser Thr 130 135140 Asn Phe Ser Cys Val Leu Val Asp Pro Glu Gln Val Val Gln Arg His 145150 155 160 Val Val Leu Ala Gln Leu Trp Ala Gly Leu Arg Ala Thr Leu ProPro 165 170 175 Thr Gln Glu Ala Leu Pro Ser Ser His Ser Ser Pro Gln GlnGln Gly 180 185 190 Arg Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys ProAla Pro Glu 195 200 205 Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro ProLys Pro Lys Asp 210 215 220 Thr Leu Met Ile Ser Arg Thr Pro Glu Val ThrCys Val Val Val Asp 225 230 235 240 Val Ser His Glu Asp Pro Glu Val LysPhe Asn Trp Tyr Val Asp Gly 245 250 255 Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Tyr Asn 260 265 270 Ser Thr Tyr Arg Val Val SerVal Leu Thr Val Leu His Gln Asp Trp 275 280 285 Leu Asn Gly Lys Glu TyrLys Cys Lys Val Ser Asn Lys Ala Leu Pro 290 295 300 Ala Pro Ile Glu LysThr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 305 310 315 320 Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 325 330 335 Gln ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 340 345 350 AlaVal Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 355 360 365Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 370 375380 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 385390 395 400 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu 405 410 415 Ser Leu Ser Pro Gly Lys 420 12 579 DNA Homo sapiens CDS(1)..(579) sig_peptide (1)..(108) mat_peptide (109)..() 12 atg gct gctgaa cca gta gaa gac aat tgc atc aac ttt gtg gca atg 48 Met Ala Ala GluPro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met -35 -30 -25 aaa ttt attgac aat acg ctt tac ttt ata gct gaa gat gat gaa aac 96 Lys Phe Ile AspAsn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn -20 -15 -10 -5 ctg gaatca gat tac ttt ggc aag ctt gaa tct aaa tta tca gtc ata 144 Leu Glu SerAsp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile -1 1 5 10 aga aatttg aat gac caa gtt ctc ttc att gac caa gga aat cgg cct 192 Arg Asn LeuAsn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro 15 20 25 cta ttt gaagat atg act gat tct gac tgt aga gat aat gca ccc cgg 240 Leu Phe Glu AspMet Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg 30 35 40 acc ata ttt attata agt atg tat aaa gat agc cag cct aga ggt atg 288 Thr Ile Phe Ile IleSer Met Tyr Lys Asp Ser Gln Pro Arg Gly Met 45 50 55 60 gct gta act atctct gtg aag tgt gag aaa att tca ayt ctc tcc tgt 336 Ala Val Thr Ile SerVal Lys Cys Glu Lys Ile Ser Xaa Leu Ser Cys 65 70 75 gag aac aaa att atttcc ttt aag gaa atg aat cct cct gat aac atc 384 Glu Asn Lys Ile Ile SerPhe Lys Glu Met Asn Pro Pro Asp Asn Ile 80 85 90 aag gat aca aaa agt gacatc ata ttc ttt cag aga agt gtc cca gga 432 Lys Asp Thr Lys Ser Asp IleIle Phe Phe Gln Arg Ser Val Pro Gly 95 100 105 cat gat aat aag atg caattt gaa tct tca tca tac gaa gga tac ttt 480 His Asp Asn Lys Met Gln PheGlu Ser Ser Ser Tyr Glu Gly Tyr Phe 110 115 120 cta gct tgt gaa aaa gagaga gac ctt ttt aaa ctc att ttg aaa aaa 528 Leu Ala Cys Glu Lys Glu ArgAsp Leu Phe Lys Leu Ile Leu Lys Lys 125 130 135 140 gag gat gaa ttg ggggat aga tct ata atg ttc act gtt caa aac gaa 576 Glu Asp Glu Leu Gly AspArg Ser Ile Met Phe Thr Val Gln Asn Glu 145 150 155 gac 579 Asp 13 193PRT Homo sapiens misc_feature (73)..(73) The ′Xaa′ at location 73 standsfor Thr, or Ile. 13 Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn PheVal Ala Met -35 -30 -25 Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala GluAsp Asp Glu Asn -20 -15 -10 -5 Leu Glu Ser Asp Tyr Phe Gly Lys Leu GluSer Lys Leu Ser Val Ile -1 1 5 10 Arg Asn Leu Asn Asp Gln Val Leu PheIle Asp Gln Gly Asn Arg Pro 15 20 25 Leu Phe Glu Asp Met Thr Asp Ser AspCys Arg Asp Asn Ala Pro Arg 30 35 40 Thr Ile Phe Ile Ile Ser Met Tyr LysAsp Ser Gln Pro Arg Gly Met 45 50 55 60 Ala Val Thr Ile Ser Val Lys CysGlu Lys Ile Ser Xaa Leu Ser Cys 65 70 75 Glu Asn Lys Ile Ile Ser Phe LysGlu Met Asn Pro Pro Asp Asn Ile 80 85 90 Lys Asp Thr Lys Ser Asp Ile IlePhe Phe Gln Arg Ser Val Pro Gly 95 100 105 His Asp Asn Lys Met Gln PheGlu Ser Ser Ser Tyr Glu Gly Tyr Phe 110 115 120 Leu Ala Cys Glu Lys GluArg Asp Leu Phe Lys Leu Ile Leu Lys Lys 125 130 135 140 Glu Asp Glu LeuGly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu 145 150 155 Asp 14 157PRT Homo sapiens 14 Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile ArgAsn Leu Asn 1 5 10 15 Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg ProLeu Phe Glu Asp 20 25 30 Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro ArgThr Ile Phe Ile 35 40 45 Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly MetAla Val Thr Ile 50 55 60 Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser CysGlu Asn Lys Ile 65 70 75 80 Ile Ser Phe Lys Glu Met Asn Pro Pro Asp AsnIle Lys Asp Thr Lys 85 90 95 Ser Asp Ile Ile Phe Phe Gln Arg Ser Val ProGly His Asp Asn Lys 100 105 110 Met Gln Phe Glu Ser Ser Ser Tyr Glu GlyTyr Phe Leu Ala Cys Glu 115 120 125 Lys Glu Arg Asp Leu Phe Lys Leu IleLeu Lys Lys Glu Asp Glu Leu 130 135 140 Gly Asp Arg Ser Ile Met Phe ThrVal Gln Asn Glu Asp 145 150 155 15 765 DNA Homo sapiens CDS (4)..(765)15 gtc atg gaa tac gcc tct gac gct tca ctg gac ccc gaa gcc ccg tgg 48Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp 1 5 10 15cct ccc gcg ccc cgc gct cgc gcc tgc cgc gta ctg cct tgg gcc ctg 96 ProPro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu 20 25 30 gtcgcg ggg ctg ctg ctg ctg ctg ctg ctc gct gcc gcc tgc gcc gtc 144 Val AlaGly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val 35 40 45 ttc ctcgcc tgc ccc tgg gcc gtg tcc ggg gct cgc gcc tcg ccc ggc 192 Phe Leu AlaCys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly 50 55 60 tcc gcg gccagc ccg aga ctc cgc gag ggt ccc gag ctt tcg ccc gac 240 Ser Ala Ala SerPro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp 65 70 75 gat ccc gcc ggcctc ttg gac ctg cgg cag ggc atg ttt gcg cag ctg 288 Asp Pro Ala Gly LeuLeu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu 80 85 90 95 gtg gcc caa aatgtt ctg ctg atc gat ggg ccc ctg agc tgg tac agt 336 Val Ala Gln Asn ValLeu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 100 105 110 gac cca ggc ctggca ggc gtg tcc ctg acg ggg ggc ctg agc tac aaa 384 Asp Pro Gly Leu AlaGly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 115 120 125 gag gac acg aaggag ctg gtg gtg gcc aag gct gga gtc tac tat gtc 432 Glu Asp Thr Lys GluLeu Val Val Ala Lys Ala Gly Val Tyr Tyr Val 130 135 140 ttc ttt caa ctagag ctg cgg cgc gtg gtg gcc ggc gag ggc tca ggc 480 Phe Phe Gln Leu GluLeu Arg Arg Val Val Ala Gly Glu Gly Ser Gly 145 150 155 tcc gtt tca cttgcg ctg cac ctg cag cca ctg cgc tct gct gct ggg 528 Ser Val Ser Leu AlaLeu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 160 165 170 175 gcc gcc gccctg gct ttg acc gtg gac ctg cca ccc gcc tcc tcc gag 576 Ala Ala Ala LeuAla Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 180 185 190 gct cgg aactcg gcc ttc ggt ttc cag ggc cgc ttg ctg cac ctg agt 624 Ala Arg Asn SerAla Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser 195 200 205 gcc ggc cagcgc ctg ggc gtc cat ctt cac act gag gcc agg gca cgc 672 Ala Gly Gln ArgLeu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 210 215 220 cat gcc tggcag ctt acc cag ggc gcc aca gtc ttg gga ctc ttc cgg 720 His Ala Trp GlnLeu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg 225 230 235 gtg acc cccgaa atc cca gcc gga ctc cct tca ccg agg tcg gaa 765 Val Thr Pro Glu IlePro Ala Gly Leu Pro Ser Pro Arg Ser Glu 240 245 250 16 254 PRT Homosapiens 16 Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro TrpPro 1 5 10 15 Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp AlaLeu Val 20 25 30 Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys AlaVal Phe 35 40 45 Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser ProGly Ser 50 55 60 Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser ProAsp Asp 65 70 75 80 Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe AlaGln Leu Val 85 90 95 Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser TrpTyr Ser Asp 100 105 110 Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly LeuSer Tyr Lys Glu 115 120 125 Asp Thr Lys Glu Leu Val Val Ala Lys Ala GlyVal Tyr Tyr Val Phe 130 135 140 Phe Gln Leu Glu Leu Arg Arg Val Val AlaGly Glu Gly Ser Gly Ser 145 150 155 160 Val Ser Leu Ala Leu His Leu GlnPro Leu Arg Ser Ala Ala Gly Ala 165 170 175 Ala Ala Leu Ala Leu Thr ValAsp Leu Pro Pro Ala Ser Ser Glu Ala 180 185 190 Arg Asn Ser Ala Phe GlyPhe Gln Gly Arg Leu Leu His Leu Ser Ala 195 200 205 Gly Gln Arg Leu GlyVal His Leu His Thr Glu Ala Arg Ala Arg His 210 215 220 Ala Trp Gln LeuThr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val 225 230 235 240 Thr ProGlu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 245 250 17 1415 DNA Homosapiens CDS (120)..(884) sig_peptide (120)..(189) mat_peptide (189)..()17 agtggaaagt tctccggcag ccctgagatc tcaagagtga catttgtgag accagctaat 60ttgattaaaa ttctcttgga atcagctttg ctagtatcat acctgtgcca gatttcatc 119 atggga aac agc tgt tac aac ata gta gcc act ctg ttg ctg gtc ctc 167 Met GlyAsn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu -20 -15 -10 aacttt gag agg aca aga tca ttg cag gat cct tgt agt aac tgc cca 215 Asn PheGlu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro -5 -1 1 5 gctggt aca ttc tgt gat aat aac agg aat cag att tgc agt ccc tgt 263 Ala GlyThr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 10 15 20 25 cctcca aat agt ttc tcc agc gca ggt gga caa agg acc tgt gac ata 311 Pro ProAsn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 30 35 40 tgc aggcag tgt aaa ggt gtt ttc agg acc agg aag gag tgt tcc tcc 359 Cys Arg GlnCys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 45 50 55 acc agc aatgca gag tgt gac tgc act cca ggg ttt cac tgc ctg ggg 407 Thr Ser Asn AlaGlu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 60 65 70 gca gga tgc agcatg tgt gaa cag gat tgt aaa caa ggt caa gaa ctg 455 Ala Gly Cys Ser MetCys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 75 80 85 aca aaa aaa ggt tgtaaa gac tgt tgc ttt ggg aca ttt aac gat cag 503 Thr Lys Lys Gly Cys LysAsp Cys Cys Phe Gly Thr Phe Asn Asp Gln 90 95 100 105 aaa cgt ggc atctgt cga ccc tgg aca aac tgt tct ttg gat gga aag 551 Lys Arg Gly Ile CysArg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 110 115 120 tct gtg ctt gtgaat ggg acg aag gag agg gac gtg gtc tgt gga cca 599 Ser Val Leu Val AsnGly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 125 130 135 tct cca gcc gacctc tct ccg gga gca tcc tct gtg acc ccg cct gcc 647 Ser Pro Ala Asp LeuSer Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 140 145 150 cct gcg aga gagcca gga cac tct ccg cag atc atc tcc ttc ttt ctt 695 Pro Ala Arg Glu ProGly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 155 160 165 gcg ctg acg tcgact gcg ttg ctc ttc ctg ctg ttc ttc ctc acg ctc 743 Ala Leu Thr Ser ThrAla Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 170 175 180 185 cgt ttc tctgtt gtt aaa cgg ggc aga aag aaa ctc ctg tat ata ttc 791 Arg Phe Ser ValVal Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 190 195 200 aaa caa ccattt atg aga cca gta caa act act caa gag gaa gat ggc 839 Lys Gln Pro PheMet Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 205 210 215 tgt agc tgccga ttt cca gaa gaa gaa gaa gga gga tgt gaa ctg 884 Cys Ser Cys Arg PhePro Glu Glu Glu Glu Gly Gly Cys Glu Leu 220 225 230 tgaaatggaagtcaataggg ctgttgggac tttcttgaaa agaagcaagg aaatatgagt 944 catccgctatcacagctttc aaaagcaaga acaccatcct acataatacc caggattccc 1004 ccaacacacgttcttttcta aatgccaatg agttggcctt taaaaatgca ccactttttt 1064 ttttttttttgacagggtct cactctgtca cccaggctgg agtgcagtgg caccaccatg 1124 gctctctgcagccttgacct ctgggagctc aagtgatcct cctgcctcag tctcctagta 1184 gctggaactacaaggaaggg ccaccacacc tgactaactt ttttgttttt tgtttggtaa 1244 agatggcatttcgccatgtt gtacaggctg gtctcaaact cctaggttca ctttggcctc 1304 ccaaagtgctgggattacag acatgaactg ccaggcccgg ccaaaataat gcaccacttt 1364 taacagaacagacagatgag gacagagctg gtgataaaaa aaaaaaaaaa a 1415 18 255 PRT Homosapiens 18 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu ValLeu -20 -15 -10 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser AsnCys Pro -5 -1 1 5 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile CysSer Pro Cys 10 15 20 25 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln ArgThr Cys Asp Ile 30 35 40 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg LysGlu Cys Ser Ser 45 50 55 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly PheHis Cys Leu Gly 60 65 70 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys GlnGly Gln Glu Leu 75 80 85 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly ThrPhe Asn Asp Gln 90 95 100 105 Lys Arg Gly Ile Cys Arg Pro Trp Thr AsnCys Ser Leu Asp Gly Lys 110 115 120 Ser Val Leu Val Asn Gly Thr Lys GluArg Asp Val Val Cys Gly Pro 125 130 135 Ser Pro Ala Asp Leu Ser Pro GlyAla Ser Ser Val Thr Pro Pro Ala 140 145 150 Pro Ala Arg Glu Pro Gly HisSer Pro Gln Ile Ile Ser Phe Phe Leu 155 160 165 Ala Leu Thr Ser Thr AlaLeu Leu Phe Leu Leu Phe Phe Leu Thr Leu 170 175 180 185 Arg Phe Ser ValVal Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 190 195 200 Lys Gln ProPhe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 205 210 215 Cys SerCys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 220 225 230 19 648DNA Homo sapiens CDS (1)..(645) 19 atg cat gtg ccg gcg ggc tcc gtg gccagc cac ctg ggg acc acg agc 48 Met His Val Pro Ala Gly Ser Val Ala SerHis Leu Gly Thr Thr Ser 1 5 10 15 cgc agc tat ttc tat ttg acc aca gccact ctg gct ctg tgc ctt gtc 96 Arg Ser Tyr Phe Tyr Leu Thr Thr Ala ThrLeu Ala Leu Cys Leu Val 20 25 30 ttc acg gtg gcc act att atg gtg ttg gtcgtt cag agg acg gac tcc 144 Phe Thr Val Ala Thr Ile Met Val Leu Val ValGln Arg Thr Asp Ser 35 40 45 att ccc aac tca cct gac aac gtc ccc ctc aaagga gga aat tgc tca 192 Ile Pro Asn Ser Pro Asp Asn Val Pro Leu Lys GlyGly Asn Cys Ser 50 55 60 gaa gac ctc tta tgt atc ctg aaa aga gct cca ttcaag aag tca tgg 240 Glu Asp Leu Leu Cys Ile Leu Lys Arg Ala Pro Phe LysLys Ser Trp 65 70 75 80 gcc tac ctc caa gtg gca aag cat cta aac aaa accaag ttg tct tgg 288 Ala Tyr Leu Gln Val Ala Lys His Leu Asn Lys Thr LysLeu Ser Trp 85 90 95 aac aaa gat ggc att ctc cat gga gtc aga tat cag gatggg aat ctg 336 Asn Lys Asp Gly Ile Leu His Gly Val Arg Tyr Gln Asp GlyAsn Leu 100 105 110 gtg atc caa ttc cct ggt ttg tac ttc atc att tgc caactg cag ttt 384 Val Ile Gln Phe Pro Gly Leu Tyr Phe Ile Ile Cys Gln LeuGln Phe 115 120 125 ctt gta caa tgc cca aat aat tct gtc gat ctg aag ttggag ctt ctc 432 Leu Val Gln Cys Pro Asn Asn Ser Val Asp Leu Lys Leu GluLeu Leu 130 135 140 atc aac aag cat atc aaa aaa cag gcc ctg gtg aca gtgtgt gag tct 480 Ile Asn Lys His Ile Lys Lys Gln Ala Leu Val Thr Val CysGlu Ser 145 150 155 160 gga atg caa acg aaa cac gta tac cag aat ctc tctcaa ttc ttg ctg 528 Gly Met Gln Thr Lys His Val Tyr Gln Asn Leu Ser GlnPhe Leu Leu 165 170 175 gat tac ctg cag gtc aac acc acc ata tca gtc aatgtg gat aca ttc 576 Asp Tyr Leu Gln Val Asn Thr Thr Ile Ser Val Asn ValAsp Thr Phe 180 185 190 cag tac ata gat aca agc acc ttt cct ctt gag aatgtg ttg tcc atc 624 Gln Tyr Ile Asp Thr Ser Thr Phe Pro Leu Glu Asn ValLeu Ser Ile 195 200 205 ttc tta tac agt aat tca gac tga 648 Phe Leu TyrSer Asn Ser Asp 210 215 20 215 PRT Homo sapiens 20 Met His Val Pro AlaGly Ser Val Ala Ser His Leu Gly Thr Thr Ser 1 5 10 15 Arg Ser Tyr PheTyr Leu Thr Thr Ala Thr Leu Ala Leu Cys Leu Val 20 25 30 Phe Thr Val AlaThr Ile Met Val Leu Val Val Gln Arg Thr Asp Ser 35 40 45 Ile Pro Asn SerPro Asp Asn Val Pro Leu Lys Gly Gly Asn Cys Ser 50 55 60 Glu Asp Leu LeuCys Ile Leu Lys Arg Ala Pro Phe Lys Lys Ser Trp 65 70 75 80 Ala Tyr LeuGln Val Ala Lys His Leu Asn Lys Thr Lys Leu Ser Trp 85 90 95 Asn Lys AspGly Ile Leu His Gly Val Arg Tyr Gln Asp Gly Asn Leu 100 105 110 Val IleGln Phe Pro Gly Leu Tyr Phe Ile Ile Cys Gln Leu Gln Phe 115 120 125 LeuVal Gln Cys Pro Asn Asn Ser Val Asp Leu Lys Leu Glu Leu Leu 130 135 140Ile Asn Lys His Ile Lys Lys Gln Ala Leu Val Thr Val Cys Glu Ser 145 150155 160 Gly Met Gln Thr Lys His Val Tyr Gln Asn Leu Ser Gln Phe Leu Leu165 170 175 Asp Tyr Leu Gln Val Asn Thr Thr Ile Ser Val Asn Val Asp ThrPhe 180 185 190 Gln Tyr Ile Asp Thr Ser Thr Phe Pro Leu Glu Asn Val LeuSer Ile 195 200 205 Phe Leu Tyr Ser Asn Ser Asp 210 215 21 705 DNA Homosapiens CDS (1)..(702) 21 atg gac cca ggg ctg cag caa gca ctc aac ggaatg gcc cct cct gga 48 Met Asp Pro Gly Leu Gln Gln Ala Leu Asn Gly MetAla Pro Pro Gly 1 5 10 15 gac aca gcc atg cat gtg ccg gcg ggc tcc gtggcc agc cac ctg ggg 96 Asp Thr Ala Met His Val Pro Ala Gly Ser Val AlaSer His Leu Gly 20 25 30 acc acg agc cgc agc tat ttc tat ttg acc aca gccact ctg gct ctg 144 Thr Thr Ser Arg Ser Tyr Phe Tyr Leu Thr Thr Ala ThrLeu Ala Leu 35 40 45 tgc ctt gtc ttc acg gtg gcc act att atg gtg ttg gtcgtt cag agg 192 Cys Leu Val Phe Thr Val Ala Thr Ile Met Val Leu Val ValGln Arg 50 55 60 acg gac tcc att ccc aac tca cct gac aac gtc ccc ctc aaagga gga 240 Thr Asp Ser Ile Pro Asn Ser Pro Asp Asn Val Pro Leu Lys GlyGly 65 70 75 80 aat tgc tca gaa gac ctc tta tgt atc ctg aaa aga gct ccattc aag 288 Asn Cys Ser Glu Asp Leu Leu Cys Ile Leu Lys Arg Ala Pro PheLys 85 90 95 aag tca tgg gcc tac ctc caa gtg gca aag cat cta aac aaa accaag 336 Lys Ser Trp Ala Tyr Leu Gln Val Ala Lys His Leu Asn Lys Thr Lys100 105 110 ttg tct tgg aac aaa gat ggc att ctc cat gga gtc aga tat caggat 384 Leu Ser Trp Asn Lys Asp Gly Ile Leu His Gly Val Arg Tyr Gln Asp115 120 125 ggg aat ctg gtg atc caa ttc cct ggt ttg tac ttc atc att tgccaa 432 Gly Asn Leu Val Ile Gln Phe Pro Gly Leu Tyr Phe Ile Ile Cys Gln130 135 140 ctg cag ttt ctt gta caa tgc cca aat aat tct gtc gat ctg aagttg 480 Leu Gln Phe Leu Val Gln Cys Pro Asn Asn Ser Val Asp Leu Lys Leu145 150 155 160 gag ctt ctc atc aac aag cat atc aaa aaa cag gcc ctg gtgaca gtg 528 Glu Leu Leu Ile Asn Lys His Ile Lys Lys Gln Ala Leu Val ThrVal 165 170 175 tgt gag tct gga atg caa acg aaa cac gta tac cag aat ctctct caa 576 Cys Glu Ser Gly Met Gln Thr Lys His Val Tyr Gln Asn Leu SerGln 180 185 190 ttc ttg ctg gat tac ctg cag gtc aac acc acc ata tca gtcaat gtg 624 Phe Leu Leu Asp Tyr Leu Gln Val Asn Thr Thr Ile Ser Val AsnVal 195 200 205 gat aca ttc cag tac ata gat aca agc acc ttt cct ctt gagaat gtg 672 Asp Thr Phe Gln Tyr Ile Asp Thr Ser Thr Phe Pro Leu Glu AsnVal 210 215 220 ttg tcc atc ttc tta tac agt aat tca gac tga 705 Leu SerIle Phe Leu Tyr Ser Asn Ser Asp 225 230 22 234 PRT Homo sapiens 22 MetAsp Pro Gly Leu Gln Gln Ala Leu Asn Gly Met Ala Pro Pro Gly 1 5 10 15Asp Thr Ala Met His Val Pro Ala Gly Ser Val Ala Ser His Leu Gly 20 25 30Thr Thr Ser Arg Ser Tyr Phe Tyr Leu Thr Thr Ala Thr Leu Ala Leu 35 40 45Cys Leu Val Phe Thr Val Ala Thr Ile Met Val Leu Val Val Gln Arg 50 55 60Thr Asp Ser Ile Pro Asn Ser Pro Asp Asn Val Pro Leu Lys Gly Gly 65 70 7580 Asn Cys Ser Glu Asp Leu Leu Cys Ile Leu Lys Arg Ala Pro Phe Lys 85 9095 Lys Ser Trp Ala Tyr Leu Gln Val Ala Lys His Leu Asn Lys Thr Lys 100105 110 Leu Ser Trp Asn Lys Asp Gly Ile Leu His Gly Val Arg Tyr Gln Asp115 120 125 Gly Asn Leu Val Ile Gln Phe Pro Gly Leu Tyr Phe Ile Ile CysGln 130 135 140 Leu Gln Phe Leu Val Gln Cys Pro Asn Asn Ser Val Asp LeuLys Leu 145 150 155 160 Glu Leu Leu Ile Asn Lys His Ile Lys Lys Gln AlaLeu Val Thr Val 165 170 175 Cys Glu Ser Gly Met Gln Thr Lys His Val TyrGln Asn Leu Ser Gln 180 185 190 Phe Leu Leu Asp Tyr Leu Gln Val Asn ThrThr Ile Ser Val Asn Val 195 200 205 Asp Thr Phe Gln Tyr Ile Asp Thr SerThr Phe Pro Leu Glu Asn Val 210 215 220 Leu Ser Ile Phe Leu Tyr Ser AsnSer Asp 225 230 23 1788 DNA Homo sapiens CDS (1)..(1785) 23 atg cgc gtcctc ctc gcc gcg ctg gga ctg ctg ttc ctg ggg gcg cta 48 Met Arg Val LeuLeu Ala Ala Leu Gly Leu Leu Phe Leu Gly Ala Leu 1 5 10 15 cga gcc ttccca cag gat cga ccc ttc gag gac acc tgt cat gga aac 96 Arg Ala Phe ProGln Asp Arg Pro Phe Glu Asp Thr Cys His Gly Asn 20 25 30 ccc agc cac tactat gac aag gct gtc agg agg tgc tgt tac cgc tgc 144 Pro Ser His Tyr TyrAsp Lys Ala Val Arg Arg Cys Cys Tyr Arg Cys 35 40 45 ccc atg ggg ctg ttcccg aca cag cag tgc cca cag agg cct act gac 192 Pro Met Gly Leu Phe ProThr Gln Gln Cys Pro Gln Arg Pro Thr Asp 50 55 60 tgc agg aag cag tgt gagcct gac tac tac ctg gat gag gcc gac cgc 240 Cys Arg Lys Gln Cys Glu ProAsp Tyr Tyr Leu Asp Glu Ala Asp Arg 65 70 75 80 tgt aca gcc tgc gtg acttgt tct cga gat gac ctc gtg gag aag acg 288 Cys Thr Ala Cys Val Thr CysSer Arg Asp Asp Leu Val Glu Lys Thr 85 90 95 ccg tgt gca tgg aac tcc tcccgt gtc tgc gaa tgt cga ccc ggc atg 336 Pro Cys Ala Trp Asn Ser Ser ArgVal Cys Glu Cys Arg Pro Gly Met 100 105 110 ttc tgt tcc acg tct gcc gtcaac tcc tgt gcc cgc tgc ttc ttc cat 384 Phe Cys Ser Thr Ser Ala Val AsnSer Cys Ala Arg Cys Phe Phe His 115 120 125 tct gtc tgt ccg gca ggg atgatt gtc aag ttc cca ggc acg gcg cag 432 Ser Val Cys Pro Ala Gly Met IleVal Lys Phe Pro Gly Thr Ala Gln 130 135 140 aag aac acg gtc tgt gag ccggct tcc cca ggg gtc agc cct gcc tgt 480 Lys Asn Thr Val Cys Glu Pro AlaSer Pro Gly Val Ser Pro Ala Cys 145 150 155 160 gcc agc cca gag aac tgcaag gaa ccc tcc agt ggc acc atc ccc cag 528 Ala Ser Pro Glu Asn Cys LysGlu Pro Ser Ser Gly Thr Ile Pro Gln 165 170 175 gcc aag ccc acc ccg gtgtcc cca gca acc tcc agt gcc agc acc atg 576 Ala Lys Pro Thr Pro Val SerPro Ala Thr Ser Ser Ala Ser Thr Met 180 185 190 cct gta aga ggg ggc acccgc ctc gcc cag gaa gct gct tct aaa ctg 624 Pro Val Arg Gly Gly Thr ArgLeu Ala Gln Glu Ala Ala Ser Lys Leu 195 200 205 acg agg gct ccc gac tctccc tcc tct gtg gga agg cct agt tca gat 672 Thr Arg Ala Pro Asp Ser ProSer Ser Val Gly Arg Pro Ser Ser Asp 210 215 220 cca ggt ctg tcc cca acacag cca tgc cca gag ggg tct ggt gat tgc 720 Pro Gly Leu Ser Pro Thr GlnPro Cys Pro Glu Gly Ser Gly Asp Cys 225 230 235 240 aga aag cag tgt gagccc gac tac tac ctg gac gag gcc ggc cgc tgc 768 Arg Lys Gln Cys Glu ProAsp Tyr Tyr Leu Asp Glu Ala Gly Arg Cys 245 250 255 aca gcc tgc gtg agctgt tct cga gat gac ctt gtg gag aag acg cca 816 Thr Ala Cys Val Ser CysSer Arg Asp Asp Leu Val Glu Lys Thr Pro 260 265 270 tgt gca tgg aac tcctcc cgc acc tgc gaa tgt cga cct ggc atg atc 864 Cys Ala Trp Asn Ser SerArg Thr Cys Glu Cys Arg Pro Gly Met Ile 275 280 285 tgt gcc aca tca gccacc aac tcc tgt gcc cgc tgt gtc ccc tac cca 912 Cys Ala Thr Ser Ala ThrAsn Ser Cys Ala Arg Cys Val Pro Tyr Pro 290 295 300 atc tgt gca gga gagacg gtc acc aag ccc cag gat atg gct gag aag 960 Ile Cys Ala Gly Glu ThrVal Thr Lys Pro Gln Asp Met Ala Glu Lys 305 310 315 320 gac acc acc tttgag gcg cca ccc ctg ggg acc cag ccg gac tgc aac 1008 Asp Thr Thr Phe GluAla Pro Pro Leu Gly Thr Gln Pro Asp Cys Asn 325 330 335 ccc acc cca gagaat ggc gag gcg cct gcc agc acc agc ccc act cag 1056 Pro Thr Pro Glu AsnGly Glu Ala Pro Ala Ser Thr Ser Pro Thr Gln 340 345 350 agc ttg ctg gtggac tcc cag gcc agt aag acg ctg ccc atc cca acc 1104 Ser Leu Leu Val AspSer Gln Ala Ser Lys Thr Leu Pro Ile Pro Thr 355 360 365 agc gct ccc gtcgct ctc tcc tcc acg ggg aag ccc gtt ctg gat gca 1152 Ser Ala Pro Val AlaLeu Ser Ser Thr Gly Lys Pro Val Leu Asp Ala 370 375 380 ggg cca gtg ctcttc tgg gtg atc ctg gtg ttg gtt gtg gtg gtc ggc 1200 Gly Pro Val Leu PheTrp Val Ile Leu Val Leu Val Val Val Val Gly 385 390 395 400 tcc agc gccttc ctc ctg tgc cac cgg agg gcc tgc agg aag cga att 1248 Ser Ser Ala PheLeu Leu Cys His Arg Arg Ala Cys Arg Lys Arg Ile 405 410 415 cgg cag aagctc cac ctg tgc tac ccg gtc cag acc tcc cag ccc aag 1296 Arg Gln Lys LeuHis Leu Cys Tyr Pro Val Gln Thr Ser Gln Pro Lys 420 425 430 cta gag cttgtg gat tcc aga ccc agg agg agc tca acg cag ctg agg 1344 Leu Glu Leu ValAsp Ser Arg Pro Arg Arg Ser Ser Thr Gln Leu Arg 435 440 445 agt ggt gcgtcg gtg aca gaa ccc gtc gcg gaa gag cga ggg tta atg 1392 Ser Gly Ala SerVal Thr Glu Pro Val Ala Glu Glu Arg Gly Leu Met 450 455 460 agc cag ccactg atg gag acc tgc cac agc gtg ggg gca gcc tac ctg 1440 Ser Gln Pro LeuMet Glu Thr Cys His Ser Val Gly Ala Ala Tyr Leu 465 470 475 480 gag agcctg ccg ctg cag gat gcc agc ccg gcc ggg ggc ccc tcg tcc 1488 Glu Ser LeuPro Leu Gln Asp Ala Ser Pro Ala Gly Gly Pro Ser Ser 485 490 495 ccc agggac ctt cct gag ccc cgg gtg tcc acg gag cac acc aat aac 1536 Pro Arg AspLeu Pro Glu Pro Arg Val Ser Thr Glu His Thr Asn Asn 500 505 510 aag attgag aaa atc tac atc atg aag gct gac acc gtg atc gtg ggg 1584 Lys Ile GluLys Ile Tyr Ile Met Lys Ala Asp Thr Val Ile Val Gly 515 520 525 acc gtgaag gct gag ctg ccg gag ggc cgg ggc ctg gcg ggg cca gca 1632 Thr Val LysAla Glu Leu Pro Glu Gly Arg Gly Leu Ala Gly Pro Ala 530 535 540 gag cccgag ttg gag gag gag ctg gag gcg gac cat acc ccc cac tac 1680 Glu Pro GluLeu Glu Glu Glu Leu Glu Ala Asp His Thr Pro His Tyr 545 550 555 560 cccgag cag gag aca gaa ccg cct ctg ggc agc tgc agc gat gtc atg 1728 Pro GluGln Glu Thr Glu Pro Pro Leu Gly Ser Cys Ser Asp Val Met 565 570 575 ctctca gtg gaa gag gaa ggg aaa gaa gac ccc ttg ccc aca gct gcc 1776 Leu SerVal Glu Glu Glu Gly Lys Glu Asp Pro Leu Pro Thr Ala Ala 580 585 590 tctgga aag tga 1788 Ser Gly Lys 595 24 595 PRT Homo sapiens 24 Met Arg ValLeu Leu Ala Ala Leu Gly Leu Leu Phe Leu Gly Ala Leu 1 5 10 15 Arg AlaPhe Pro Gln Asp Arg Pro Phe Glu Asp Thr Cys His Gly Asn 20 25 30 Pro SerHis Tyr Tyr Asp Lys Ala Val Arg Arg Cys Cys Tyr Arg Cys 35 40 45 Pro MetGly Leu Phe Pro Thr Gln Gln Cys Pro Gln Arg Pro Thr Asp 50 55 60 Cys ArgLys Gln Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Asp Arg 65 70 75 80 CysThr Ala Cys Val Thr Cys Ser Arg Asp Asp Leu Val Glu Lys Thr 85 90 95 ProCys Ala Trp Asn Ser Ser Arg Val Cys Glu Cys Arg Pro Gly Met 100 105 110Phe Cys Ser Thr Ser Ala Val Asn Ser Cys Ala Arg Cys Phe Phe His 115 120125 Ser Val Cys Pro Ala Gly Met Ile Val Lys Phe Pro Gly Thr Ala Gln 130135 140 Lys Asn Thr Val Cys Glu Pro Ala Ser Pro Gly Val Ser Pro Ala Cys145 150 155 160 Ala Ser Pro Glu Asn Cys Lys Glu Pro Ser Ser Gly Thr IlePro Gln 165 170 175 Ala Lys Pro Thr Pro Val Ser Pro Ala Thr Ser Ser AlaSer Thr Met 180 185 190 Pro Val Arg Gly Gly Thr Arg Leu Ala Gln Glu AlaAla Ser Lys Leu 195 200 205 Thr Arg Ala Pro Asp Ser Pro Ser Ser Val GlyArg Pro Ser Ser Asp 210 215 220 Pro Gly Leu Ser Pro Thr Gln Pro Cys ProGlu Gly Ser Gly Asp Cys 225 230 235 240 Arg Lys Gln Cys Glu Pro Asp TyrTyr Leu Asp Glu Ala Gly Arg Cys 245 250 255 Thr Ala Cys Val Ser Cys SerArg Asp Asp Leu Val Glu Lys Thr Pro 260 265 270 Cys Ala Trp Asn Ser SerArg Thr Cys Glu Cys Arg Pro Gly Met Ile 275 280 285 Cys Ala Thr Ser AlaThr Asn Ser Cys Ala Arg Cys Val Pro Tyr Pro 290 295 300 Ile Cys Ala GlyGlu Thr Val Thr Lys Pro Gln Asp Met Ala Glu Lys 305 310 315 320 Asp ThrThr Phe Glu Ala Pro Pro Leu Gly Thr Gln Pro Asp Cys Asn 325 330 335 ProThr Pro Glu Asn Gly Glu Ala Pro Ala Ser Thr Ser Pro Thr Gln 340 345 350Ser Leu Leu Val Asp Ser Gln Ala Ser Lys Thr Leu Pro Ile Pro Thr 355 360365 Ser Ala Pro Val Ala Leu Ser Ser Thr Gly Lys Pro Val Leu Asp Ala 370375 380 Gly Pro Val Leu Phe Trp Val Ile Leu Val Leu Val Val Val Val Gly385 390 395 400 Ser Ser Ala Phe Leu Leu Cys His Arg Arg Ala Cys Arg LysArg Ile 405 410 415 Arg Gln Lys Leu His Leu Cys Tyr Pro Val Gln Thr SerGln Pro Lys 420 425 430 Leu Glu Leu Val Asp Ser Arg Pro Arg Arg Ser SerThr Gln Leu Arg 435 440 445 Ser Gly Ala Ser Val Thr Glu Pro Val Ala GluGlu Arg Gly Leu Met 450 455 460 Ser Gln Pro Leu Met Glu Thr Cys His SerVal Gly Ala Ala Tyr Leu 465 470 475 480 Glu Ser Leu Pro Leu Gln Asp AlaSer Pro Ala Gly Gly Pro Ser Ser 485 490 495 Pro Arg Asp Leu Pro Glu ProArg Val Ser Thr Glu His Thr Asn Asn 500 505 510 Lys Ile Glu Lys Ile TyrIle Met Lys Ala Asp Thr Val Ile Val Gly 515 520 525 Thr Val Lys Ala GluLeu Pro Glu Gly Arg Gly Leu Ala Gly Pro Ala 530 535 540 Glu Pro Glu LeuGlu Glu Glu Leu Glu Ala Asp His Thr Pro His Tyr 545 550 555 560 Pro GluGln Glu Thr Glu Pro Pro Leu Gly Ser Cys Ser Asp Val Met 565 570 575 LeuSer Val Glu Glu Glu Gly Lys Glu Asp Pro Leu Pro Thr Ala Ala 580 585 590Ser Gly Lys 595 25 696 DNA Homo sapiens CDS (138)..(686) 25 ggccctgggacctttgccta ttttctgatt gataggcttt gttttgtctt tacctccttc 60 tttctggggaaaacttcagt tttatcgcac gttccccttt tccatatctt catcttccct 120 ctacccagattgtgaag atg gaa agg gtc caa ccc ctg gaa gag aat gtg 170 Met Glu Arg ValGln Pro Leu Glu Glu Asn Val 1 5 10 gga aat gca gcc agg cca aga ttc gagagg aac aag cta ttg ctg gtg 218 Gly Asn Ala Ala Arg Pro Arg Phe Glu ArgAsn Lys Leu Leu Leu Val 15 20 25 gcc tct gta att cag gga ctg ggg ctg ctcctg tgc ttc acc tac atc 266 Ala Ser Val Ile Gln Gly Leu Gly Leu Leu LeuCys Phe Thr Tyr Ile 30 35 40 tgc ctg cac ttc tct gct ctt cag gta tca catcgg tat cct cga att 314 Cys Leu His Phe Ser Ala Leu Gln Val Ser His ArgTyr Pro Arg Ile 45 50 55 caa agt atc aaa gta caa ttt acc gaa tat aag aaggag aaa ggt ttc 362 Gln Ser Ile Lys Val Gln Phe Thr Glu Tyr Lys Lys GluLys Gly Phe 60 65 70 75 atc ctc act tcc caa aag gag gat gaa atc atg aaggtg cag aac aac 410 Ile Leu Thr Ser Gln Lys Glu Asp Glu Ile Met Lys ValGln Asn Asn 80 85 90 tca gtc atc atc aac tgt gat ggg ttt tat ctc atc tccctg aag ggc 458 Ser Val Ile Ile Asn Cys Asp Gly Phe Tyr Leu Ile Ser LeuLys Gly 95 100 105 tac ttc tcc cag gaa gtc aac att agc ctt cat tac cagaag gat gag 506 Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu His Tyr Gln LysAsp Glu 110 115 120 gag ccc ctc ttc caa ctg aag aag gtc agg tct gtc aactcc ttg atg 554 Glu Pro Leu Phe Gln Leu Lys Lys Val Arg Ser Val Asn SerLeu Met 125 130 135 gtg gcc tct ctg act tac aaa gac aaa gtc tac ttg aatgtg acc act 602 Val Ala Ser Leu Thr Tyr Lys Asp Lys Val Tyr Leu Asn ValThr Thr 140 145 150 155 gac aat acc tcc ctg gat gac ttc cat gtg aat ggcgga gaa ctg att 650 Asp Asn Thr Ser Leu Asp Asp Phe His Val Asn Gly GlyGlu Leu Ile 160 165 170 ctt atc cat caa aat cct ggt gaa ttc tgt gtc ctttgaggggctg 696 Leu Ile His Gln Asn Pro Gly Glu Phe Cys Val Leu 175 18026 183 PRT Homo sapiens 26 Met Glu Arg Val Gln Pro Leu Glu Glu Asn ValGly Asn Ala Ala Arg 1 5 10 15 Pro Arg Phe Glu Arg Asn Lys Leu Leu LeuVal Ala Ser Val Ile Gln 20 25 30 Gly Leu Gly Leu Leu Leu Cys Phe Thr TyrIle Cys Leu His Phe Ser 35 40 45 Ala Leu Gln Val Ser His Arg Tyr Pro ArgIle Gln Ser Ile Lys Val 50 55 60 Gln Phe Thr Glu Tyr Lys Lys Glu Lys GlyPhe Ile Leu Thr Ser Gln 65 70 75 80 Lys Glu Asp Glu Ile Met Lys Val GlnAsn Asn Ser Val Ile Ile Asn 85 90 95 Cys Asp Gly Phe Tyr Leu Ile Ser LeuLys Gly Tyr Phe Ser Gln Glu 100 105 110 Val Asn Ile Ser Leu His Tyr GlnLys Asp Glu Glu Pro Leu Phe Gln 115 120 125 Leu Lys Lys Val Arg Ser ValAsn Ser Leu Met Val Ala Ser Leu Thr 130 135 140 Tyr Lys Asp Lys Val TyrLeu Asn Val Thr Thr Asp Asn Thr Ser Leu 145 150 155 160 Asp Asp Phe HisVal Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn 165 170 175 Pro Gly GluPhe Cys Val Leu 180 27 865 DNA Homo sapiens CDS (15)..(845) 27cagcagagac gagg atg tgc gtg ggg gct cgg cgg ctg ggc cgc ggg ccg 50 MetCys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro 1 5 10 tgt gcg gct ctg ctcctc ctg ggc ctg ggg ctg agc acc gtg acg ggg 98 Cys Ala Ala Leu Leu LeuLeu Gly Leu Gly Leu Ser Thr Val Thr Gly 15 20 25 ctc cac tgt gtc ggg gacacc tac ccc agc aac gac cgg tgc tgc cac 146 Leu His Cys Val Gly Asp ThrTyr Pro Ser Asn Asp Arg Cys Cys His 30 35 40 gag tgc agg cca ggc aac gggatg gtg agc cgc tgc agc cgc tcc cag 194 Glu Cys Arg Pro Gly Asn Gly MetVal Ser Arg Cys Ser Arg Ser Gln 45 50 55 60 aac acg gtg tgc cgt ccg tgcggg ccg ggc ttc tac aac gac gtg gtc 242 Asn Thr Val Cys Arg Pro Cys GlyPro Gly Phe Tyr Asn Asp Val Val 65 70 75 agc tcc aag ccg tgc aag ccc tgcacg tgg tgt aac ctc aga agt ggg 290 Ser Ser Lys Pro Cys Lys Pro Cys ThrTrp Cys Asn Leu Arg Ser Gly 80 85 90 agt gag cgg aag cag ctg tgc acg gccaca cag gac aca gtc tgc cgc 338 Ser Glu Arg Lys Gln Leu Cys Thr Ala ThrGln Asp Thr Val Cys Arg 95 100 105 tgc cgg gcg ggc acc cag ccc ctg gacagc tac aag cct gga gtt gac 386 Cys Arg Ala Gly Thr Gln Pro Leu Asp SerTyr Lys Pro Gly Val Asp 110 115 120 tgt gcc ccc tgc cct cca ggg cac ttctcc cca ggc gac aac cag gcc 434 Cys Ala Pro Cys Pro Pro Gly His Phe SerPro Gly Asp Asn Gln Ala 125 130 135 140 tgc aag ccc tgg acc aac tgc accttg gct ggg aag cac acc ctg cag 482 Cys Lys Pro Trp Thr Asn Cys Thr LeuAla Gly Lys His Thr Leu Gln 145 150 155 ccg gcc agc aat agc tcg gac gcaatc tgt gag gac agg gac ccc cca 530 Pro Ala Ser Asn Ser Ser Asp Ala IleCys Glu Asp Arg Asp Pro Pro 160 165 170 gcc acg cag ccc cag gag acc cagggc ccc ccg gcc agg ccc atc act 578 Ala Thr Gln Pro Gln Glu Thr Gln GlyPro Pro Ala Arg Pro Ile Thr 175 180 185 gtc cag ccc act gaa gcc tgg cccaga acc tca cag gga ccc tcc acc 626 Val Gln Pro Thr Glu Ala Trp Pro ArgThr Ser Gln Gly Pro Ser Thr 190 195 200 cgg ccc gtg gag gtc ccc ggg ggccgt gcg gtt gcc gcc atc ctg ggc 674 Arg Pro Val Glu Val Pro Gly Gly ArgAla Val Ala Ala Ile Leu Gly 205 210 215 220 ctg ggc ctg gtg ctg ggg ctgctg ggc ccc ctg gcc atc ctg ctg gcc 722 Leu Gly Leu Val Leu Gly Leu LeuGly Pro Leu Ala Ile Leu Leu Ala 225 230 235 ctg tac ctg ctc cgg agg gaccag agg ctg ccc ccc gat gcc cac aag 770 Leu Tyr Leu Leu Arg Arg Asp GlnArg Leu Pro Pro Asp Ala His Lys 240 245 250 ccc cct ggg gga ggc agt ttccgg acc ccc atc caa gag gag cag gcc 818 Pro Pro Gly Gly Gly Ser Phe ArgThr Pro Ile Gln Glu Glu Gln Ala 255 260 265 gac gcc cac tcc acc ctg gccaag atc tgacctgggc ccaccaaggt 865 Asp Ala His Ser Thr Leu Ala Lys Ile270 275 28 277 PRT Homo sapiens 28 Met Cys Val Gly Ala Arg Arg Leu GlyArg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu SerThr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn AspArg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg CysSer Arg Ser Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe TyrAsn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp CysAsn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr GlnAsp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp SerTyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His PheSer Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys ThrLeu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser SerAsp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 GlnGlu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200205 Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210215 220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu225 230 235 240 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro ProGly Gly 245 250 255 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala AspAla His Ser 260 265 270 Thr Leu Ala Lys Ile 275 29 1599 DNA Homo sapiensCDS (67)..(1596) 29 ccacaccaag cagcggctgg gggggggaaa gacgaggaaagaggaggaaa acaaaagctg 60 ctactt atg gaa gat aca aag gag tct aac gtg aagaca ttt tgc tcc 108 Met Glu Asp Thr Lys Glu Ser Asn Val Lys Thr Phe CysSer 1 5 10 aag aat atc cta gcc atc ctt ggc ttc tcc tct atc ata gct gtgata 156 Lys Asn Ile Leu Ala Ile Leu Gly Phe Ser Ser Ile Ile Ala Val Ile15 20 25 30 gct ttg ctt gct gtg ggg ttg acc cag aac aaa gca ttg cca gaaaac 204 Ala Leu Leu Ala Val Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn35 40 45 gtt aag tat ggg att gtg ctg gat gcg ggt tct tct cac aca agt tta252 Val Lys Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu 5055 60 tac atc tat aag tgg cca gca gaa aag gag aat gac aca ggc gtg gtg300 Tyr Ile Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val 6570 75 cat caa gta gaa gaa tgc agg gtt aaa ggt cct gga atc tca aaa ttt348 His Gln Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe 8085 90 gtt cag aaa gta aat gaa ata ggc att tac ctg act gat tgc atg gaa396 Val Gln Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu 95100 105 110 aga gct agg gaa gtg att cca agg tcc cag cac caa gag aca cccgtt 444 Arg Ala Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val115 120 125 tac ctg gga gcc acg gca ggc atg cgg ttg ctc agg atg gaa agtgaa 492 Tyr Leu Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu130 135 140 gag ttg gca gac agg gtt ctg gat gtg gtg gag agg agc ctc agcaac 540 Glu Leu Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser Asn145 150 155 tac ccc ttt gac ttc cag ggt gcc agg atc att act ggc caa gaggaa 588 Tyr Pro Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu Glu160 165 170 ggt gcc tat ggc tgg att act atc aac tat ctg ctg ggc aaa ttcagt 636 Gly Ala Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys Phe Ser175 180 185 190 cag aaa aca agg tgg ttc agc ata gtc cca tat gaa acc aataat cag 684 Gln Lys Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu Thr Asn AsnGln 195 200 205 gaa acc ttt gga gct ttg gac ctt ggg gga gcc tct aca caagtc act 732 Glu Thr Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln ValThr 210 215 220 ttt gta ccc caa aac cag act atc gag tcc cca gat aat gctctg caa 780 Phe Val Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn Ala LeuGln 225 230 235 ttt cgc ctc tat ggc aag gac tac aat gtc tac aca cat agcttc ttg 828 Phe Arg Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser PheLeu 240 245 250 tgc tat ggg aag gat cag gca ctc tgg cag aaa ctg gcc aaggac att 876 Cys Tyr Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys AspIle 255 260 265 270 cag gtt gca agt aat gaa att ctc agg gac cca tgc tttcat cct gga 924 Gln Val Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe HisPro Gly 275 280 285 tat aag aag gta gtg aac gta agt gac ctt tac aag accccc tgc acc 972 Tyr Lys Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr ProCys Thr 290 295 300 aag aga ttt gag atg act ctt cca ttc cag cag ttt gaaatc cag ggt 1020 Lys Arg Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu IleGln Gly 305 310 315 att gga aac tat caa caa tgc cat caa agc atc ctg gagctc ttc aac 1068 Ile Gly Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu LeuPhe Asn 320 325 330 acc agt tac tgc cct tac tcc cag tgt gcc ttc aat gggatt ttc ttg 1116 Thr Ser Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly IlePhe Leu 335 340 345 350 cca cca ctc cag ggg gat ttt ggg gca ttt tca gctttt tac ttt gtg 1164 Pro Pro Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala PheTyr Phe Val 355 360 365 atg aag ttt tta aac ttg aca tca gag aaa gtc tctcag gaa aag gtg 1212 Met Lys Phe Leu Asn Leu Thr Ser Glu Lys Val Ser GlnGlu Lys Val 370 375 380 act gag atg atg aaa aag ttc tgt gct cag cct tgggag gag ata aaa 1260 Thr Glu Met Met Lys Lys Phe Cys Ala Gln Pro Trp GluGlu Ile Lys 385 390 395 aca tct tac gct gga gta aag gag aag tac ctg agtgaa tac tgc ttt 1308 Thr Ser Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser GluTyr Cys Phe 400 405 410 tct ggt acc tac att ctc tcc ctc ctt ctg caa ggctat cat ttc aca 1356 Ser Gly Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly TyrHis Phe Thr 415 420 425 430 gct gat tcc tgg gag cac atc cat ttc att ggcaag atc cag ggc agc 1404 Ala Asp Ser Trp Glu His Ile His Phe Ile Gly LysIle Gln Gly Ser 435 440 445 gac gcc ggc tgg act ttg ggc tac atg ctg aacctg acc aac atg atc 1452 Asp Ala Gly Trp Thr Leu Gly Tyr Met Leu Asn LeuThr Asn Met Ile 450 455 460 cca gct gag caa cca ttg tcc aca cct ctc tcccac tcc acc tat gtc 1500 Pro Ala Glu Gln Pro Leu Ser Thr Pro Leu Ser HisSer Thr Tyr Val 465 470 475 ttc ctc atg gtt cta ttc tcc ctg gtc ctt ttcaca gtg gcc atc ata 1548 Phe Leu Met Val Leu Phe Ser Leu Val Leu Phe ThrVal Ala Ile Ile 480 485 490 ggc ttg ctt atc ttt cac aag cct tca tat ttctgg aaa gat atg gta 1596 Gly Leu Leu Ile Phe His Lys Pro Ser Tyr Phe TrpLys Asp Met Val 495 500 505 510 tag 1599 30 510 PRT Homo sapiens 30 MetGlu Asp Thr Lys Glu Ser Asn Val Lys Thr Phe Cys Ser Lys Asn 1 5 10 15Ile Leu Ala Ile Leu Gly Phe Ser Ser Ile Ile Ala Val Ile Ala Leu 20 25 30Leu Ala Val Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys 35 40 45Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile 50 55 60Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln 65 70 7580 Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln 85 9095 Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala 100105 110 Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu115 120 125 Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu GluLeu 130 135 140 Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser AsnTyr Pro 145 150 155 160 Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly GlnGlu Glu Gly Ala 165 170 175 Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu GlyLys Phe Ser Gln Lys 180 185 190 Thr Arg Trp Phe Ser Ile Val Pro Tyr GluThr Asn Asn Gln Glu Thr 195 200 205 Phe Gly Ala Leu Asp Leu Gly Gly AlaSer Thr Gln Val Thr Phe Val 210 215 220 Pro Gln Asn Gln Thr Ile Glu SerPro Asp Asn Ala Leu Gln Phe Arg 225 230 235 240 Leu Tyr Gly Lys Asp TyrAsn Val Tyr Thr His Ser Phe Leu Cys Tyr 245 250 255 Gly Lys Asp Gln AlaLeu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val 260 265 270 Ala Ser Asn GluIle Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys 275 280 285 Lys Val ValAsn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290 295 300 Phe GluMet Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly 305 310 315 320Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser 325 330335 Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro 340345 350 Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys355 360 365 Phe Leu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu Lys Val ThrGlu 370 375 380 Met Met Lys Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile LysThr Ser 385 390 395 400 Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser Glu TyrCys Phe Ser Gly 405 410 415 Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly TyrHis Phe Thr Ala Asp 420 425 430 Ser Trp Glu His Ile His Phe Ile Gly LysIle Gln Gly Ser Asp Ala 435 440 445 Gly Trp Thr Leu Gly Tyr Met Leu AsnLeu Thr Asn Met Ile Pro Ala 450 455 460 Glu Gln Pro Leu Ser Thr Pro LeuSer His Ser Thr Tyr Val Phe Leu 465 470 475 480 Met Val Leu Phe Ser LeuVal Leu Phe Thr Val Ala Ile Ile Gly Leu 485 490 495 Leu Ile Phe His LysPro Ser Tyr Phe Trp Lys Asp Met Val 500 505 510 31 8 PRT Artificialsequence Flag peptide 31 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 32 11 PRTArtificial Sequence linker 32 Gly Gly Gly Gly Ser Gly Gly Gly Gly GlySer 1 5 10 33 13 PRT Artificial sequence linker 33 Gly Ala Gly Gly AlaGly Ser Gly Gly Gly Gly Gly Ser 1 5 10 34 10 PRT Artificial sequencelinker 34 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 35 9 PRTArtificial sequence peptide 35 Gly Thr Pro Gly Thr Pro Gly Thr Pro 1 536 26 PRT Artificial Sequence peptide 36 Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly GlyGly Gly Ser 20 25

What is claimed is:
 1. A method of treating cardiovascular disease in asubject having cardiovascular disease, the method comprisingadministering to the subject a therapeutically effective amount of anantagonist, wherein the antagonist is selected from the group consistingof an IL-17 antagonist, IL-18 antagonist, 4-1BB antagonist, CD30antagonist and an OX40 antagonist.
 2. The method of claim 1, wherein theIL-17 antagonist is a soluble IL-17 receptor.
 3. The method of claim 2,wherein the soluble IL-17 receptor is a fusion protein.
 4. The method ofclaim 1, wherein the IL-17 antagonist is an antibody.
 5. The method ofclaim 4, wherein the antibody specifically binds the IL-17 receptor. 6.The method of claim 4, wherein the antibody specifically binds IL-17. 7.The method of claim 4, wherein the antibody is a humanized antibody. 8.The method of claim 4, wherein the antibody is a single-chain antibody.9. The method of claim 1, wherein the IL-17 antagonist is administeredone or more times per week.
 10. The method of claim 1, wherein the IL-17antagonist is administered by subcutaneous injection.
 11. The method ofclaim 1, wherein the IL-17 antagonist is administered in combinationwith one or more compounds selected from the group consisting ofnon-steroidal anti-inflammatory drugs; analgesics; systemic steroids;antagonists of inflammatory cytokines; anti-inflammatory cytokines;chemotherapeutics; lipid-lowering drugs; blood pressure-regulatingdrugs; angiotensin-converting enzyme inhibitors and/or peroxisomeproliferator-activated receptor ligands.